Acid sphingomyelinase protein and methods of treating type B Niemann-Pick disease

ABSTRACT

The present invention relates to the acid sphingomyelinase gene and to methods of diagnosing Niemann-Pick disease. It is based, at least in part, on the cloning and expression of the full-length cDNA encoding acid sphingomyelinase and on the discovery of mutations in the acid sphingomyelinase gene of Ashkenazi Jewish Niemann-Pick disease patients.

1. INTRODUCTION

The present invention relates to the acid sphingomyelinase gene and tomethods of diagnosing Niemann-Pick disease. It is based, at least inpart, on the cloning and expression of the full-length cDNA encodingacid sphingomyelinase, the cloning and characterization of the genomicstructure of the acid sphingomyelinase gene, and on the discovery of afrequent missense mutation in the acid sphingomyelinase gene ofAshkenazi Jewish Niemann-Pick disease patients.

2. BACKGROUND OF THE INVENTION

Types A and B Niemann-Pick disease (NPD) are autosomal recessivedisorders resulting from the deficient activity of the lysosomalhydrolase, acid sphingomyelinase (ASM; sphingomyelincholinephosphohydrolase, E:C 3.1.3.12) and the accumulation ofsphingomyelin, primarily in reticuloendothelial lysosomes (Niemann,1914, Fahrb. Kinderheikd, 79:1-6; Brady et al., 1966, Proc. Natl. Acad.Sci. U.S.A. 55;366-369; Fredrickson, 1966, in “The Metabolic Basis ofInherited Disease; Stanbury et al., eds., 2nd Ed., McGraw-Hill, NewYork, pp. 586-602; Spence and Callahan, 1989, in “The Metabolic Basis ofInherited Disease,” Scriver et al., eds., 8th Ed., McGraw-Hill, NewYork, pp. 1655-1676). Type A disease is a rapidly progressiveneurodegenerative disease of infancy manifested by failure to thrive,severe psychomotor retardation, hepatosplenomegaly, and demise by 2-3years of age. In comparison, type B disease is characterized primarilyby reticuloendothelial system sphingomyelin deposition leading tohepatosplenomegaly and pulmonary involvement, the absence of neurologicmanifestations, and survival into adulthood. The nature of thebiochemical and molecular defects that underlie the remarkable clinicalheterogeneity of the A and B subtypes remains unknown. Although patientswith both subtypes have residual ASM activity (about 1 to 10% ofnormal), biochemical analysis cannot reliably distinguish the twophenotypes. Moreover, the clinical course of Type B NPD is highlyvariable, and it is not presently possible to correlate disease severitywith the level of residual ASM activity.

Types A and B NPD occur at least 10 times more frequently amongindividuals of Ashkenazi Jewish ancestry than in the general population.It is estimated that the incidence of the type A disease among AshkenaziJews is about 1 in 40,000, a gene frequency (q) of about 1 in 200, and aheterozygote frequency (2 pq) of 1 in 100 (Goodman, 1979, in “GeneticDisorders Among The Jewish People”, John Hopkins Univ. Press, Baltimore,pp. 96-100). The incidence of type B NPD in the Ashkenazi Jewishpopulation is less frequent, perhaps 1 in 80 (Goodman, supra). Thus, thecombined heterozygotic frequency for types A and B NPD has beenestimated to be about 1 in 70 among individuals of Ashkenazi Jewishdecent. Although the enzymatic diagnosis of affected patients witheither type A or B NPD can be made reliably (Spence and Callahan,supra), the enzymatic detection of obligate heterozygotes has provenproblematic, particularly using peripheral leukocytes as the enzymesource. Presumably, the occurrence of neutral sphingomyelinases in somesources and/or the presence of residual ASM activity resulting from themutant allele have contributed to the inability to reliably discriminatecarriers for either disease subtype. Even the use of cultured skinfibroblasts, which do not express the neutral sphingomyelinase, has notprovided unambiguous results with obligate heterozygotes.

Recently, two partial cDNAs encoding human ASM were isolated andsequenced (Quintern et al., 1989, EMBO J. 8:2469-2473). The type 1 cDNAcontained an in-frame 172 base pairs (bp) encoding 57 amino acids; inthe type 2 cDNA this sequence was replaced by an in-frame 40 bp encoding13 different amino acids. Of the 92 positive clones identified by cDNAlibrary screening, the type 1 and 2 cDNAs represented about 90% and 10%,respectively (Quintern et al., supra).

3. SUMMARY OF THE INVENTION

The present invention relates to the ASM gene and to methods ofdiagnosing Niemann-Pick disease (NPD). It is based, at least in part, onthe cloning and characterization of full-length cDNAs corresponding tothree ASM gene transcripts and the recognition that one species oftranscript could be expressed to form the active ASM enzyme. The presentinvention is further based on the discovery of a frequent missensemutation in the ASM gene that was detected in 32 percent of theAshkenazi Jewish NPD type A alleles but in only 5.6 percent of ASMalleles from non-Jewish type A patients, and the discovery of a deletionmutant of the ASM gene that is associated with NPD type B disease.Additionally, the genomic sequence and structure of the ASM gene iselucidated herein.

The present invention provides for nucleic acid encoding ASM,substantially purified ASM protein and fragments and derivativesthereof, expression systems for producing ASM, genetically engineeredcells and organisms containing a recombinant full-length ASM gene,probes that may be used to diagnose mutations in ASM, assay systems forthe diagnosis of NPD, and methods of treatment of NPD.

In one preferred embodiment of the invention, such an assay system maybe used to determine the presence of a mutation that results in anarginine to leucine substitution at amino acid residue 496 in AshkenaziJewish NPD patients and in prenatal diagnosis, said mutation beingassociated with NPD type A.

In another preferred embodiment of the invention, such an assay systemmay be used to determine the presence of a mutation that results in adeletion of an arginine residue at amino acid position 608 in AshkenaziJewish NPD patients and in prenatal diagnosis, said mutation beingassociated with NPD type B.

4. DESCRIPTION OF THE FIGURES

FIG. 1. Northern hybridization and RNase protection analysis of humanASM transcripts in placenta. For the Northern hybridizations (A), about3 μg of poly(A) human placental RNA was electrophoresed and hybridizedwith the radiolabeled pASM-1 cDNA insert. The sizes of the RNA molecularmass standards are indicated; the bottom arrow identifies the single 2.5kb hybridizing transcript. The probe designed for the RNA protectionexperiment is shown schematically. The expected, protected fragment in atype 1 transcript was 333 bp, whereas in a type 2 transcript theprotected fragment would be 266 bp. Lane 1, mock control with noplacental RNA; lanes 2 (16-h exposure) and 3 (48-h exposure) containsabout 1 μg of human placental RNA. The arrows indicate the type 1 and 2protected fragments.

FIGS. 2A-2C. PCR amplification of type 2-specific transcripts from humanplacenta. Sense and antisense PCR primers (indicated by the arrows) wereconstructed from the full-length type 1 cDNA (pASM-1FL) and the partialtype 2 cDNA (pASM-2) sequences, respectively (2C). Following PCRamplification, the products were electrophoresed in 1% agarose gels (2A)and then hybridized with an ASM-specific oligonucleotide (2B). A 1.1 kbproduct was identified, consistent with the occurrence of a full-lengthtype 2 transcript in placenta.

FIGS. 3A-3C. Nucleotide (SEQ ID NO: 1) and predicted amino acid (SEQ IDNO:2) sequences of the full-length ASM cDNA, pASM-1FL. The pASM-1FLinsert was sequenced in both orientations. The unique 172-bp type 1sequence is bracketed. Underlined amino acid residues represent residuesthat were colinear with the amino acid sequences from tryptic peptidesof the purified enzyme (T-1 to T-12). The boxed amino acid residues arethose that were different from the fibroblast cDNAs, pASM-1 and pASM-2.CHO represents potential N-glycosylation sites. FIG. 4. Schematicrepresentation of human ASM type 1, 2 and 3 cDNAs. The longest type 1, 2and 3 cDNAs isolated by library screening are shown schematically. Thetype 1- and 2-specific sequences are indicated (172 and 40 bp,respectively), as are the locations of the stop codons. A and B arecommon 5′ and 3′ coding sequences, respectively. Note that the partialtype 3 cDNA has a premature termination codon (TAA).

FIG. 5. Reconstruction of full-length type 2 and 3 human ASM cDNAs. Thefull-length type 2 (A) and 3 (B) cDNAs were reconstructed as describedunder “Methods.” The 172- and 40-bp type 1- and 2-specific sequences areindicated, as are the flanking BstEII restriction sites.

FIGS. 6A-6B. Sequence of the PCR amplified genomic region containing theunique type 1- and 2-specific human ASM regions (SEQ ID NO: 3). PCRamplification of human genomic DNA was performed using primers 1 and 2.Upper and lower case letters indicate exonic and intronic sequences,respectively. The type 1- and 2-specific genomic sequences are shown inboldface type. Boxed sequences D1 to D4 and A1 and A2 indicate 5′ donorand 3′ acceptor splice site sequences, respectively. The potentiallariat branch point consensus sequences are underlined and designateda-c.

FIGS. 6C-6H. Nucleotide sequence (SEQ ID NO: 4) of the genomic regionencoding human ASM. Upper and lower case letters denote ASM exonic andintronic sequences, respectively. The two potential initiation codons inexon 1 are indicated by a double underline. The type 1-specific regionis encoded by exon 3 and the type 2-specific region at the 5′ end ofintron 2 is underlined. A potential cryptic donor splice site adjacentto the type 2-specific region is indicated by an overline. An Sp 1binding site and an Alu 1 homology region are boxed. The initiationcodons for ORFs 1, 2 and 3 also are underlined and the transcriptionaldirections are indicated by arrows.

FIG. 7. Proposed model for alternative splicing of ASM transcripts. Type1 transcripts result from normal splicing events, whereas type 2 RNAsoccur due to a single splice which brings together the 3′ acceptor, A2(nt 1590-1591), with the cryptic 5′ donor site, D3 (nt 179-180). Thus,the type 2 transcript deletes the 172 bp exon which is replaced by 40in-frame intronic bp. The type 3 transcript results from a splicingevent which joins the 3′ acceptor, A2, with the 5′ donor, D1 (nt139-140).

FIG. 8. Partial sequence of the amplified ASM cDNA from an AshkenaziJewish type A NPD homozygote (proband 1) showing the G-T transversion ofnt 1487. cDNA synthesis, PCR amplification, and DNA sequencing aredescribed. Arrows indicate the G-T transversion in proband 1 (Right)that results in R496L.

FIG. 9. Identification of R496L (SEQ ID NOS: 5 and 6), in amplifiedgenomic DNAs from the members of an Ashkenazi Jewish family with type ANPD by dot blot hybridization with allele-specific oligonucleotides(ASOs). Note that the affected homozygote (proband 1: o/) washomoallelic, and both of her parents were heterozygous for R496L.

FIG. 10. Identification of a three base deletion in the ASM genomic DNAfrom proband 2. (a) The methods for PCR amplification of the ASM genomicDNA from proband 2, subcloning of the PCR products and DNA sequencingare described in the text. A small area of the genomic sequence obtainedfrom a normal individual (left) (SEQ ID NOS: 7 and 8) and proband 2(right) (SEQ ID NOS: 9 and 10) is shown. (b) A schematic representationof the ΔR608 mutation. FIG. 11. Genotype analysis of proband 2 andfamily members by dot-blot hybridization. The conditions used fordob-blot hybridization of PCR-amplified genomic DNA with the R496L andΔR608 ASOs are described in the text.

5. DETAILED DESCRIPTION OF THE INVENTION

For purposes of clarity, and not by way of limitation, the detaileddescription of the invention is divided into the following subsections:

(i) the ASM gene;

(ii) expression of the ASM gene;

(iii) identification of mutations in the ASM gene;

(iv) methods of diagnosing Niemann-Pick disease;

(v) assay systems for diagnosing Niemann-Pick disease; and

(vi) methods of treatment of Niemann-Pick disease.

5.1. The Acid Sphingomyelinase Gene

The nucleotide coding sequence and deduced amino acid sequence for thefull-length cDNA that encodes functional ASM is depicted in FIGS. 3A-3C(SEQ ID NOS: 1 and 2), and is contained in plasmid pASM-1FL. Thisnucleotide sequence, or fragments or functional equivalents thereof, maybe used to generate recombinant DNA molecules that direct the expressionof the enzyme product, or functionally active peptides or functionalequivalents thereof, in appropriate host cells. The genomic nucleotidesequence of ASM, its characterization and structural organization isdepicted in FIGS. 6C-6H (SEQ ID NO: 4).

Due to the degeneracy of the nucleotide coding sequence, other DNAsequences which encode substantially the same amino acid sequences asdepicted in FIG. 3 may be used in the practice of the invention for thecloning and expression of ASM. Such alterations include deletions,additions or substitutions of different nucleotide residues resulting ina sequence that encodes the same or a functionally equivalent geneproduct. The gene product may contain deletions, additions orsubstitutions of amino acid residues within the sequence, which resultin a silent change thus producing a bioactive product. Such amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, the amphipathicnature of the residues involved and/or on the basis of crystallographicdata. For example, negatively charged amino acids include aspartic acidand glutamic acid; positively charged amino acids include lysine andarginine; amino acids with uncharged polar head groups having similarhydrophilicity values include the following: leucine, isoleucine,valine; glycine, alanine; asparagine, glutamine; serine, threonine;phenylalanine, tyrosine.

The coding sequences for ASM may be conveniently obtained fromgenetically engineered microorganisms or cell lines containing theenzyme coding sequences. Alternatively, genomic sequences or cDNA codingsequences for these enzymes may be obtained from human genomic or cDNAlibraries. Either genomic or cDNA libraries may be prepared from DNAfragments generated from human cell sources. The fragments which encodeASM may be identified by screening such libraries with a nucleotideprobe that is substantially complementary to any portion of the sequencedepicted in FIGS. 3A-3C (SEQ ID NOS: 1) or FIGS. 6C-6H (SEQ ID NO: 4).Indeed, sequences generated by polymerase chain reaction can be ligatedto form the full-length sequence. Although portions of the codingsequences may be utilized, full length clones, i.e., those containingthe entire coding region for ASM, may be preferable for expression.Alternatively, the coding sequences depicted in FIGS. 3A-3C may bealtered by the addition of sequences that can be used to increase levelsof expression and/or to facilitate purification.

Techniques well-known to those skilled in the art for the isolation ofDNA, generation of appropriate restriction fragments, construction ofclones and libraries, and screening recombinants may be used. For areview of such techniques, see, for example, Sambrook, et al., 1989,Molecular Cloning A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press,N.Y., Chapters 1-18.

In an alternate embodiment of the invention, the coding sequence ofFIGS. 3A-3C (SEQ ID NO: 1) could be synthesized in whole or in part,using chemical methods well-known in the art. See, for example,Caruthers, et. al., 1980, Nuc. Acids Res. Symp. Ser. 7:215-233; Crea &Horn, 1980, Nuc. Acids Res. 9(10):2331; Matteucchi & Carruthers, 1980,Tetrahedron Letters 21:719; and Chow and Kempe, 1981, Nuc. Acids Res.9(12):2807-2817.

Alternatively, the protein itself could be produced using chemicalmethods to synthesize the amino acid sequence depicted in FIGS. 3A-3C(SEQ ID NO: 2) in whole or in part. The present invention provides forsubstantially purified ASM, preferably having a sequence substantiallyas depicted in FIGS. 3A-3C, or a portion thereof that is immunogenic orbiologically active. For example, peptides can be synthesized by solidphase techniques, cleaved from the resin and purified by preparativehigh performance liquid chromatograph. (E.g., see, Creighton, 1983,Proteins, Structures and Molecular Principles, W. H. Freeman & Co., N.Y.pp. 50-60). The composition of the synthetic peptides may be confirmedby amino acid analysis or sequencing (e.g., the Edman degradationprocedure; see Creighton, 1983, Proteins, Structures and MolecularPrinciples, W. H. Freeman & Co., N.Y., pp. 34-49).

Also, the 5′ untranslated and coding regions of the nucleotide sequencecould be altered to improve the translational efficiency of the ASMmRNA.

In addition, based on X-ray crystallographic data, sequence alterationscould be undertaken to improve protein stability, e.g., introducingdisulfide bridges at the appropriate positions, and/or deleting orreplacing amino acids that are predicted to cause protein instability.These are only examples of modifications that can be engineered into theASM enzyme to produce a more active or stable protein, more enzymeprotein, or even change the catalytic specificity of the enzyme.

The present invention further provides for organisms containing thefunctional ASM gene. In various embodiments, such organisms include, butare not limited to, bacteria, yeast, eukaryotic cells, or transgenicanimals. Transgenic animals whose own ASM genes have been “knocked out”by homologous recombination and replaced with mutant ASM genes may beused as models of NPD in humans.

5.2. Expression of the Acid Sphingomyelinase Gene

In order to express a biologically active ASM, the coding sequence forthe enzyme, a functional equivalent, or a modified sequence, asdescribed in Section 5.1., supra, is inserted into an appropriateexpression vector, i.e., a vector which contains the necessary elementsfor transcription and translation of the inserted coding sequence inappropriate host cells. Host cell expression systems which possess thecellular machinery and elements for the proper processing, i.e., signalcleavage, glycosylation, phosphorylation and protein sorting arepreferred. For example, mammalian host cell expression systems arepreferred for the expression of biologically active enzymes that areproperly folded and processed; when administered in humans suchexpression products should exhibit proper tissue targeting and noadverse immunological reaction.

5.2.1. Construction of Expression Vectors and Preparation ofTransfectants

Methods which are well-known to those skilled in the art can be used toconstruct expression vectors containing the ASM coding sequence andappropriate transcriptional/translational control signals. These methodsinclude in vitro recombination/genetic recombination. See, for example,the techniques described in maniatis et al., 1982, Molecular Cloning ALaboratory Manual, Cold spring Harbor Laboratory, N.Y., Chapter 12.

In bacterial systems a number of expression vectors may beadvantageously selected depending upon the use intended for the ASMprotein expressed. For example, when large quantities of ASM are to beproduced, vectors which direct the expression of high levels of fusionprotein products that are readily purified may be desirable. Suchvectors include but are not limited to the E. coli expression vectorpUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the ASM codingsequence may be ligated into the vector in frame with the lac Z codingregion so that a hybrid AS-lac Z protein is produced; pIN vectors(Inouye & Inouye, 1985, Nucleic acids Res. 13:3101-3109; Van Heeke &Schuster, 1989, J. Biol. Chem. 264:5503-5509); and the like.

A variety of eukaryotic host-expression systems may be utilized toexpress the ASM coding sequence. Although prokaryotic systems offer thedistinct advantage of ease of manipulation and low cost of scale-up,their major drawback in the expression of ASM is their lack of properpost-translational modifications of expressed mammalian proteins.Eukaryotic systems, and preferably mammalian expression systems, allowfor proper modification to occur. Eukaryotic cells which possess thecellular machinery for proper processing of the primary transcript,glycosylation, phosphorylation, and, advantageously secretion of thegene product should be used as host cells for the expression of ASM.Mammalian cell lines are preferred. Such host cell lines may include butare not limited to CHO, VERO, BHK, HeLa, COS, MDCK, −293, WI38, etc.Appropriate eukaryotic expression vectors should be utilized to directthe expression of ASM in the host cell chosen. For example, at least twobasic approaches may be followed for the design of vectors on SV40. Thefirst is to replace the SV40 early region with the gene of interestwhile the second is to replace the late region (Hammarskjold, et al.,1986, Gene 43: 41). Early and late region replacement vectors can alsobe complemented in vitro by the appropriate SV40 mutant lacking theearly or late region. Such complementation will produce recombinantswhich are packaged into infectious capsids and which contain the ASMgene. A permissive cell line can then be infected to produce therecombinant protein. SV40-based vectors can also be used in transientexpression studies, where best results are obtained when they areintroduced into COS (CV-1, origin of SV40) cells, a derivative of CV-1(green monkey kidney cells) which contain a single copy of an origindefective SV40 genome integrated into the chromosome. These cellsactively synthesize large T antigen (SV40), thus initiating replicationfrom any plasmid containing an SV40 origin of replication.

In addition to SV40, almost every molecularly cloned virus or retrovirusmay used as a cloning or expression vehicle. Viral vectors based on anumber of retroviruses (avian and murine), adenoviruses, vaccinia virus(Cochran, et al., 1985, Proc. Natl. Acad. Sci. USA 82: 19) and polyomavirus may be used for expression. Other cloned viruses, such as JC(Howley, et al., 1980, J. Virol 36: 878), BK and the human papillomaviruses (Heilman, et al., 1980, J. Virol 36: 395), offer the potentialof being used as eukaryotic expression vectors. For example, when usingadenovirus expression vectors the ASM coding sequence may be ligated toan adenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the human enzyme in infected hosts (e.g., see Logan & Shenk,1984, Proc. Natl. Acad. Sci. (USA) 81:3655-3659). Alternatively, thevaccinia virus 7.5K promoter may be used. (e.g., see, Mackett et al.,1982, Proc. Natl. Acad. Sci. (USA) 79:7415-7419; Mackett et al., 1984,J. Virol. 49:857-864; Panicali et al., 1982, Proc. Natl. Acad. Sci.79:4927-4931). Of particular interest are vectors based on bovinepapilloma virus (Sarver, et al., 1981, Mol. Cell. Biol. 1:486). Thesevectors have the ability to replicate as extrachromosomal elements.Shortly after entry of this DNA into mouse cells, the plasmid replicatesto about 100 to 200 copies per cell. Transcription of the inserted cDNAdoes not require integration of the plasmid into the host's chromosome,thereby yielding a high level of expression. These vectors can be usedfor stable expression by including a selectable marker in the plasmid,such as the neo gene. High level expression may also be achieved usinginducible promoters such as the metallothionine IIA promoter, heat shockpromoters, etc.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, following the introduction offoreign DNA, engineered cells may be allowed to grow for 1-2 days in anenriched media, and then are switched to a selective media. Rather thanusing expression vectors which contain viral origins of replication,host cells can be transformed with the ATN or DNA controlled byappropriate expression control elements (e.g., promoter, enhancer,sequences, transcription terminators, polyadenylation sites, etc.), anda selectable marker. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. A number of selectionsystems may be used, including but not limited to the herpes simplexvirus thymidine kinase (Wigler, et al., 1977, Cell 11:223),hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski,1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes can beemployed in tk⁻, hgprt⁻ or aprt⁻ cells respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler, et al., 1980,Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc. Natl. Acad.Sci. USA 78: 1527); gpt, which confers resistance to mycophenolic acid(Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072; neo, whichconfers resistance to the aminoglycoside G-418 (Colberre-Garapin, etal., 1981, J. Mol. Biol. 150:1); and hygro, which confers resistance tohygromycin (Santerre, et al., 1984, Gene 30: 147) genes. Recently,additional selectable genes have been described, namely trpB, whichallows cells to utilize indole in place of tryptophan; hisD, whichallows cells to utilize histinol in place of histidine (Hartman &Mulligan, 1988, Proc. Natl. Acad. Sci. USA 85:8047); and ODC (ornithinedecarboxylase) which confers resistance to the ornithine decarboxylaseinhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue L., 1987,In: Current Communications in Molecular Biology, Cold Spring HarborLaboratory ed.).

Alternative eukaryotic expression systems which may be used to expressthe ASM enzymes are yeast transformed with recombinant yeast expressionvectors containing the ASM coding sequence; insect cell systems infectedwith recombinant virus expression vectors (e.g., baculovirus) containingthe ASM coding sequence; or plant cell systems infected with recombinantvirus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobaccomosaic virus, TMV) or transformed with recombinant plasmid expressionvectors (e.g., Ti plasm id) containing the ASM coding sequence.

In yeast, a number of vectors containing constitutive or induciblepromoters may be used. For a review see, Current Protocols in MolecularBiology, Vol. 2, 1988, Ed. Ausubel et al., Greene Publish. Assoc. &Wiley Interscience, Ch. 13; Grant et al., 1987, Expression and SecretionVectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 31987,Acad. Press, N.Y., Vol. 153, pp.516-544, Glover, 1986, DNA Cloning, Vol.II, IRL Press, Wash., D.C., Ch. 3; and Bitter, 1987, Heterologous GeneExpression in Yeast, Methods in Enzymology, Eds. Berger & Kimmel, Acad.Press, N.Y., Vol. 152, pp. 673-684; and The Molecular Biology of theYeast Saccharomyces, 1982, Eds. Strathern et al., Cold Spring HarborPress, Vols. I and II. For complementation assays in yeast, cDNAs forASM may be cloned into yeast episomal plasmids (YEp) which replicateautonomously in yeast due to the presence of the yeast 2μ circle. ThecDNA may be cloned behind either a constitutive yeast promoter such asADH or LEU2 or an inducible promoter such as GAL (Cloning in Yeast,Chpt. 3, R. Rothstein In: DNA Cloning Vol.11, A Practical Approach, Ed.D M Glover, 1986, IRL Press, Wash., D.C.). Constructs may contain the 5′and 3′ non-translated regions of the cognate ASM mRNA or thosecorresponding to a yeast gene. YEp plasmids transform at high efficiencyand the plasmids are extremely stable. Alternatively, vectors may beused which promote integration of foreign DNA sequences into the yeastchromosome.

In cases where plant expression vectors are used, the expression of theASM coding sequence may be driven by any of a number of promoters. Forexample, viral promoters such as the 35S RNA and 19S RNA promoters ofCAMV (Brisson et al., 1984, Nature 310:511-514), or the coat proteinpromoter of TMV (Takamatsu et al., 1987, EMBO J. 6:307-311) may be used;alternatively, plant promoters such as the small subunit of RUBISCO(Coruzzi et al., 1984, EMBO J. 3:1671-1680; Broglie et al., 1984,Science 224:838-843); or heat shock promoters, e.g., soybean hsp17.5-Eor hsp17.3-B (Gurley et al., 1986, Mol. Cell. Biol. 6:559-565) may beused. These constructs can be introduced into plant cells using Tiplasmids, Ri plasmids, plant virus vectors; direct DNA transformation;microinjection, electroporation, etc. For reviews of such techniquessee, for example, Weissbach & Weissbach, 1988, Methods for PlantMolecular Biology, Academic Press, NY, Section VIII, pp. 421-463; andGrierson & Corey, 1988, Plant Molecular Biology, 2d Ed., Blackie,London, Ch. 7-9.

An alternative expression system which could be used to express ASM isan insect system. In one such system, Autographa californica nuclearpolyhedrosis virus (ACNPV) is used as a vector to express foreign genes.The virus grows in. Spodoptera frugiperda cells. The ASM sequence may becloned into non-essential regions (for example the polyhedrin gene) ofthe virus and placed under control of an AcNPV promoter (for example thepolyhedrin promoter). Successful insertion of the coding sequence willresult in inactivation of the polyhedrin gene and production ofnon-occluded recombinant virus (i.e., virus lacking the proteinaceouscoat coded for by the polyhedrin gene). These recombinant viruses arethen used to infect Spodoptera frugiperda cells in which the insertedgene is expressed. (E.g., see Smith et al., 1983, J. Viol. 46:584;Smith, U.S. Pat. No. 4,215,051).

5.2.2. Identification of Transfectants or Transformants Expressing theAsh Product

The host cells which contain the ASM coding sequence and which expressthe biologically active gene product may be identified by at least fourgeneral approaches: (a) DNA-DNA or DNA-RNA hybridization; (b) thepresence or absence of “marker” gene functions; (c) assessing the levelof transcription as measured by the expression of ASM mRNA transcriptsin the host cell; and (d) detection of the gene product as measured byimmunoassay or by its biological activity.

In the first approach, the presence of the ASM coding sequence insertedin the expression vector can be detected by DNA-DNA or DNA-RNAhybridization using probes comprising nucleotide sequences that arehomologous to the ASM coding sequence substantially as shown in FIGS.3A-3C (SEQ ID NO: 1), or portions or derivatives thereof.

In the second approach, the recombinant expression vector/host systemcan be identified and selected based upon the presence or absence ofcertain “marker” gene functions (e.g., thymidine kinase activity,resistance to antibiotics, resistance to methotrexate, transformationphenotype, occlusion body formation in baculovirus, etc.). For example,if the ASM coding sequence is inserted within a marker gene sequence ofthe vector, recombinants containing the ASM coding sequence can beidentified by the absence of the marker gene function. Alternatively, amarker gene can be placed in tandem with the ASM sequence under thecontrol of the same or different promoter used to control the expressionof the ASM coding sequence. Expression of the marker in response toinduction or selection indicates expression of the ASM coding sequence.

In the third approach, transcriptional activity for the ASM codingregion can be assessed by hybridization assays. For example, RNA can beisolated and analyzed by Northern blot using a probe homologous to theASM coding sequence or particular portions thereof substantially asshown in FIGS. 3A-3C (SEQ ID NO: 1). Alternatively, total nucleic acidsof the host cell may be extracted and assayed for hybridization to suchprobes.

In the fourth approach, the expression of the ASM protein product can beassessed immunologically, for example, by Western blots, immunoassayssuch as radioimmunoprecipitation, enzyme-linked immunoassays and thelike. The ultimate test of the success of the expression system,however, involves the detection of the biologically active ASM geneproduct. Where the host cell secretes the gene product, the cell freemedia obtained from the cultured transfectant host cell may be assayedfor ASM activity. Where the gene product is not secreted, cell lysatesmay be assayed for such activity. In either case, a number of assays canbe used to detect ASM activity including but not limited to assaysemploying N-12[(12 pyrenesulfonyl amidododecanoyl)]-sphingomyelin(PAS₁₂) (Klar et al., 1988, Clin. Chem. Acta 176:259-268), or othersubstrates for ASM.

5.2.3. Purification of the Ash Gene Product

Once a clone that produces high levels of biologically active ASM isidentified, the clone may be expanded and used to produce large amountsof the enzyme which may be purified using techniques well-known in theart including, but not limited to, immunoaffinity purification,chromatographic methods including high performance liquid chromatographyand the like. Where the enzyme is secreted by the cultured cells, ASMmay be readily recovered from the culture medium.

Where the ASM coding sequence is engineered to encode a cleavable fusionprotein, the purification of ASM may be readily accomplished usingaffinity purification techniques.

5.3. Identification of Mutations of the Acid Sphingomyelinase Gene

The present invention also provides for methods of identifying mutationsof the ASM gene. Such mutations include but are not limited tosubstitutions, insertions, or deletions in the nucleic acid or aminoacid sequence of the ASM gene. According to these embodiments, nucleicacid probes derived from known ASM genes may be used to identify mutantASM sequences in DNA or RNA obtained from a human subject suspected ofcarrying an ASM mutation. Such probes may be used in standardhybridization procedures for screening genomic or cDNA libraries (e.g.Benton and Davis, 1977, Science 196:180), or may be used in proceduresthat amplify mutant sequences, including polymerase chain reaction (PCR;Saiki et al., 1985, Science 230:1350-1354).

For example, and not by way of limitation, PCR may be used to identifymutations in the ASM gene as follows. Total RNA and genomic DNA may beprepared from cells (including cell lines) derived from a personsuspected of carrying an ASM mutation using standard techniques(Sambrook et al., 1989, in “Molecular Cloning: A Laboratory Manual,”Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). First-strandcDNA may be reverse-transcribed from about 5 μg of total RNA by using acDNA synthesis kit according to the manufacturer's instructions (e.g.,Boehringer Mannheim). The cDNA (about 10% of the total reaction) orgenomic DNA (about 0.5 μg) may be amplified by PCR using Thermusaguaticus (Tag) polymerase, essentially as described by Saiki, supra,with the following conditions and modifications. PCR may desirably beperformed for 30 to 40 cycles consisting of denaturation for 1 min. at94 degrees C. and hybridization and extension for 4 min. at 66 degreesC. To improve the specificity of the PCR amplification, a “PCR boost”procedure may be used. In this procedure, the concentrations of theprimers and Taq polymerase may be about 0.1 μM and 5 units/ml,respectively, for the first 15 cycles. Then each primer may be added toa final concentration of about 0.5 μM, and an additional 2 units of Taqpolymerase may be added to the reaction mixture. PCR amplification maythen be continued for an additional 15-25 cycles. For use as primers inthe PCR, pairs of sense and antisense primers may be prepared by anymethod known in the art, including synthesis on. an Applied Biosystemsmodel 380B DNA synthesizer (Itakura et al., 1984, Annu. Rev. Biochem.53:323-356) and used to specifically amplify, for example, either (i)the entire coding region of reverse-transcribed type 1 ASM transcript inthree overlapping cDNA fragments and/or (ii) the 1665-bp genomic regioncontaining the alternatively spliced sequences in type 1 and 2 ASMcDNAs. For these purposes, the following primers may be used:

(i) to amplify the coding region of reverse-transcribed type 1 ASMtranscript, (a) a 984 bp fragment from the 5′ end may be amplified usingthe 29-mer sense primer, P1 (5′AGTAGTCTCGAGACGGGACAGACGAACCA-3′) (SEQ IDNO: 11), corresponding to ASM nucleotide −39 to −23 with an additional12 nucleotides that include an XhoI restriction site, and the 31-merantisense primer, P2 (5′-AGTAGTCTGCAGAGCAGGGTACATGGCACTG-3′) (SEQ ID NO:12), corresponding to ASM nucleotide 926 to 945 with an additional 12nucleotides containing a HindIII restriction site. To amplify aninternal 383-bp fragment of the ASM cDNA, the 29-mer sense primer, P3(5′-ATCATCAAGCTTGGGTAACCATGAAAGCA-3′) (SEQ ID NO: 13) may be usedcorresponding to ASM nucleotides 947-964 with an additional 12nucleotides containing a HindIII restriction site, and the antisense32-mer primer, P4 (5′-ATCATCGAATTCTACAATTCGGTAATAATTCC-3′) (SEQ ID NO:14), corresponding to ASM nucleotides 1310 to 1330 with an additional 12nucleotides containing an EcoR1 restriction site. To amplify a 789 bp 3′fragment from ASM cDNA, a 19-mer sense primer, P5(5′-CTCCACGGATCCCGCAGGA-3′) (SEQ ID NO: 15), corresponding to ASMnucleotides 1185 to 1203 and containing an internal BamHI restrictionsite may be used together with an antisense 32-mer primer, P6(5′-AGTAGTGTCGACTTGCCTGGTTGAACCACAGC-3′) (SEQ ID NO: 16) correspondingto ASM nucleotides 1955 to 1974 with an additional 12 nucleotidescontaining a SalI restriction site;

(ii) to amplify the 1665 base pair genomic region containing thealternatively spliced sequences in type 1 and 2 ASM cDNAS, primers P3and P4 (supra) may be used.

The products of PCR may then be subcloned into an appropriate vectorsuch as, for example, Bluescript KS (+) (Stratagene, La Jolla, Calif.)or pGEM 9ZF (−) (Promega, Madison, Wis.). For each amplified product, itmay be desirable to sequence multiple (e.g., four to ten) independentsubclones, by methods known in the art, including, but not limited to,the dideoxy method (Sanger et al., 1977, Proc. Natl. Acad. Sci. U.S.A.,74:5463-5467). The sequences thus obtained may be compared to thesequence of ASM type 1 as set forth in FIG. 3 in order to identifymutations.

The use of this method in identifying mutations in ASM is exemplified inSections 7 and 8, infra, which relate to the Arg-Leu substitution atresidue 496 (SEQ ID NO: 34) and to the Arg deletion at residue 608 (SEQID NO: 35), respectively. The present invention provides nucleic acidscomprising these mutations, as set forth in FIG. 8 (SEQ ID NO: 6) andFIG. 10 (SEQ ID NO: 9), respectively, and primers which may be used toidentify these mutations (see infra).

5.4. Methods of Diagnosing Niemann-Pick Disease

The present invention also provides for a method of diagnosingNiemann-Pick disease (NPD) in a patient, comprising detecting a mutationin an ASM gene, in a nucleic acid sample of the patient in which themutation positively correlates with NPD. Such methods may beparticularly useful when they distinguish NPD type A from NPD type B,because the two types of disease carry clinically very differentprognoses. Such a distinction may be made by detecting the presence orabsence of an ASM mutation which is selectively associated with type Aor type B disease. Such mutations, in particular embodiments, comprisean alteration of at least one amino acid in the sequence set forth inFIGS. 3A-3C (SEQ ID NO: 2).

The methods of diagnosis of the invention may be used to diagnose NPD ina patient that is suspected of suffering from NPD or, alternatively, ina patient that is a fetus by prenatal diagnosis, using techniques suchas amniocentesis or chorionic villi sampling or by any technique knownin the art.

In related embodiments, the present invention also provides for a methodof identifying a person as having the potential to genetically transmitNPD comprising detecting a mutation in an ASM gene in a nucleic acidsample of the person, in which the mutation positively correlates withthe ability to genetically transmit NPD. A nucleic acid sample may beobtained from any suitable source of the individual's cells or tissues.In such embodiments, it may again be preferable to distinguish thepotential for transmitting NPD type A versus type B.

It should be noted that persons capable of transmitting NPD but who donot themselves suffer from NPD are likely to be heterozygotes withrespect to the ASM gene; that is, they carry one mutant and one normalgene. Persons that suffer from NPD are likely to lack a normal ASM genealtogether and to instead carry mutations in the ASM gene in both number11 chromosomes, although these mutant genes need not be the same.

Mutations in the ASM gene may be detected by the methods set forth inSection 5.3., supra, for cloning and identifying new mutations.Alternatively, mutations that have already been identified andcharacterized using the methods set forth in Section 5.3 may bedetected. For example, and not by way of limitation, the arginine toleucine mutation at amino acid residue 496 of ASM (the R496L mutation)NPD type A allele (see Section 7, infra), or the arginine deletion atamino acid 608 (the ΔR608 mutation) allele associated with type B NPD(see Section 8, infra), may be detected in methods of diagnosing NPD orof identifying a person as being capable of genetically transmitting NPDand in particular NPD type A or type B. Such probes may be particularlyuseful when analyzing the genetic material from persons of AshkenaziJewish descent.

The presence of a mutation may be detected by any method known in theart, including cloning and sequencing the ASM gene from the person to betested. In preferred methods of the invention, the presence of themutation is detected by amplifying the nucleic acids spanning themutation within the ASM gene, using sense and antisense oligonucleotideprimers designed to span the area of the ASM gene that contains themutation. For example, in specific embodiments of the invention, nucleicacid collected from the person to be tested (prepared from tissue,cells, blood, amniotic fluid or other body fluids, etc.) may be utilizedin PCR as described in Section 5.3, supra, using the following primerpairs. To detect the R496L mutation or the ΔR608 mutation, a 27-mersense primer, P7 (5′-AGTAGTCGACATGGGCAGGATGTGTGG-3′) (SEQ ID NO: 17) maybe used together with antisense primer P6 (see supra) to amplify a 567bp genomic fragment containing the G-T transversion. The resultingproducts of PCR may then be sequenced or, preferably, be analyzed forthe ability to hybridize to a normal ASO or an oligonucleotidecontaining a defined mutation. In this specific example, the R496Lmutation can be detected by hybridizing the PCR amplified sample toeither the normal oligonucleotide P8 (5′-CTATTTGGTACACACGG-3′) (SEQ IDNO: 18) or the mutation-specific oligonucleotide P9(5′-CTATTTGGTACACAAGG-3′) (SEQ ID NO: 19), in which selectivehybridization to P9 positively correlates with the presence of the R496Lmutation. Similarly, the ΔR608 mutation can be detected by hybridizingthe PCR amplified sample to either the P10 normal oligonucleotide(5′-CTCTGTGCCGCCACCTG-3′) (SEQ ID NO: 20) or the mutation-specificoligonucleotide P11 (5′-GCTCTGTGCCACCTGAT-3′) (SEQ ID NO: 21) (seeSections 7 and 8, infra).

In specific embodiments of the invention, such hybridization may be doneusing dot-blot hybridization (Sambrook et al., supra), using, forexample, Zetabind nylon membranes (AMF Cuno) and Bio-Rad dot-blotapparatus. Hybridization may be performed for at least 3 hours at 30degrees C. After hybridization, the blots may be washed at roomtemperature for 15 minutes in 6×SSC (1×SSC is 0.15 M sodiumchloride/0.015 M sodium citrate, pH 7.0)/0.1% SDS and then for 2 hoursin the same solution at either about 53-54 degrees C. for the normalallele or about 48-50 degrees C. for the mutant allele.

With regard to the R496L mutation, it was detected in 32% (10 of 31) ofAshkenazi Jewish NPD type A alleles studied; in only 5.6% (2 of 36) ofASM alleles from non-Jewish type A patients, in one of two AshkenaziJewish NPD type B patients, and in none of 180 ASM alleles from normalindividuals of Ashkenazi Jewish descent. It therefore appears that theR496L mutation results in neuronopathic type A disease when homoallelicand nonneuronopathic type B phenotype when heteroallelic with a type Bmutation such as TR608. In contrast, the ΔR608 mutation appears to occurfrequently in Type B NPD patients of Ashkenazi Jewish descent.

5.5. Assay Systems for Diagnosing Niemann-Pick Disease

The present invention also provides for kits and assay systems that maybe used in the methods described in Section 5.4, supra. Such kitscomprise oligonucleotide primers that may be used to identify mutationsin the ASM gene.

The following kits are specific, non-limiting embodiments of theinvention.

(i) A kit for identifying new mutations in the ASM gene comprisingoligonucleotide primers P1, P2, P3, P4, P5 and P6 (supra);

(ii) A kit for detecting the R496L or ΔR608 mutation in the ASM genecomprising oligonucleotide primers P6 and P7 (supra);

(iii) A kit for detecting the R496L mutation consisting of the kit in(ii), supra, and further comprising oligonucleotide hybridization probesP8 and P9 (supra);

(iv) A kit for detecting the ΔR608 mutation consisting of the kit in(ii) supra, and further comprising oligonucleotide hybridization probesP10 and P11 (supra).

5.6. Methods of Treatment of Niemann-Pick Disease

The present invention also provides for methods of treatment of NPD,particularly type B disease, comprising administering to a patient inneed of such treatment an effective amount of substantially purified ASMtype 1, prepared as described supra, or a derivative thereof. Forexample, the recombinant enzyme could be administered at a dosageranging from 0.1 mg/kg to about 10 mg/kg and, preferably from about 0.1mg/kg to about 2 mg/kg. The ability to produce large amounts of therecombinant enzyme or substantially pure enzyme in accordance with thisinvention will permit the evaluation of the therapeutic effect ofsignificantly larger doses.

Alternatively, treatment can be provided by administering to a patientin need of such treatment an effective amount of nucleic acid encodingfunctional ASM type 1, e.g., the having sequence set forth in FIGS.3A-3C (SEQ ID NO: 1). Such nucleic acid may be administered via asuitable vector, including retroviral or other viral vectors, or may beadministered via cells transfected with ASM type-1 encoding nucleicacid.

5.7. Engineering Transgenic Animals Containing the Human ASM Gene

The gene sequence of ASM is disclosed herein. The cDNA sequence of FIGS.3A-3C (SEQ ID NO: 1) or the genomic sequence of FIGS. 6C-6H (SEQ ID NO:4) can be engineered in transgenic animals to produce a model system forstudying the synthesis and regulation of the ASM enzyme. In particular,animals containing the various mutations disclosed herein can beengineered to study the effects of the mutation on the synthesis,regulation and function of ASM. Any technique known to those skilled inthe art can be used to produce the transgenic animals.

The engineering of transgenic mice containing the wild type gene isdescribed in Section 6.2.7. infra.

6. Example: Isolation Nucleotide Sequence, and Expression of the FullLength and Alternatively Spliced cDNAS Encoding Human AcidSphingomyelinase 6.1. Materials and Methods 6.1.1. Materials

Normal human placental tissue was frozen at −70° C. within 30 minutes ofdelivery and stored until use. λgt11 human placental, testis, andhepatoma cDNA libraries were obtained from Clontech Laboratories (PaloAlto, Calif.). A λgt11 human retinal cDNA library was kindly provided byDr. Jeremy Nathans (Johns Hopkins University, Baltimore, Md.).Restriction endonucleases, T4 DNA ligase, T4 polynucleotide kinase, theKlenow fragment of DNA polymerase 1, RNA molecular weight markers, andcDNA synthesis kits were obtained from New England Biolabs (Beverly,Mass.) and/or from Boehringer Mannheim. Taq polymerase was purchasedfrom Perkin-Elmer Cetus Instruments, and Sequenase DNA sequencing kitswere from U.S. Biochemical Corp. Bluescript vectors and helper phage,RNA transcription kits, Proteinase K, and RNase-free DNase 1 wereobtained from Stratagene (La Jolla, Calif.). RNase T1 was from BethesdaResearch Laboratories. Nitrocellulose (type HATF) and nylon membraneswere purchased from Millipore (Bedford, Mass.). Reagents for DNAsynthesis were obtained from Applied Biosystems (Foster City Calif.).Radioactive nucleotides and multiprime DNA labeling kits were fromAmersham Corp. N-12[(1-Pyrenesulfonyl)amidododecanoyl]-sphingomyelin wasa gift from Dr. Shimon Gatt, Hebrew University-Hadassah Medical Center(Jerusalem, Israel). The eukaryotic expression vector p91023(B) wasobtained from Dr. Randal Kaufman, Genetics Institute (Boston, Mass.).All other reagents were the highest grade available from commercialsources.

6.1.2. Northern Hybridization, RNase Protection Analyses, and Type2-Specific PCR Amplification

Total cellular RNA from human placenta (˜5 g) was prepared by amodification of the guanidine isothiocyanate procedure (Chirgwin et al.,1979, Biochemistry 18:5294-5299), and poly(A)⁺ RNA was isolated byoligo(dT)-cellulose chromatography. Aliquots of total (˜10 μg) andpoly(A)⁺ RNA (˜3 μg) RNAs were analyzed by electrophoresis throughdenaturing formaldehyde-agarose gels. Northern hybridizations wereperformed by standard techniques (Thomas, 1980, Proc. Natl. Acad. Sci.U.S.A. 77:5201-5205) using the radiolabeled pASM-1 insert as a probe.RNase protection experiments were performed according to the method ofZinn et al. (Zinn et al., 1983, Cell 34:865-879). A 333-bp BamHI-SacIrestriction fragment isolated from pASM-1FL was subcloned into the SK(+)Bluescript vector in order to prepare the radiolabeled riboprobe. AfterRNase treatment, the protected fragments were electrophoresed in 6.0%denaturing polyacrylamide gels, and the intensity of the autoradiogramwas determined by densitometry. For the amplification of type 2-specificcDNAs, a type 2-specific antisense oligonucleotide(5′-ATCATTGAATTCCACGGACGATAAGTAC -3′) (SEQ ID NO: 22) was used with asense oligonucleotide (5′-ATCATCCTCGAGACGGGACAGACGAACCA-3′) (SEQ ID NO:23) constructed from the 5′ end of the pASM-1FL cDNA insert. For thetemplate, cDNA was prepared from total placental RNA using a cDNAsynthesis kit according to the manufacturer's instructions.Alternatively, the pASM-1FL insert was used as a template to demonstratethe type 2 specificity of the PCR amplification.

6.1.3. cDNA Library Screening and Isolation of the Full-Length HumanType 1 ASM cDNA

For library screenings, human placental, testis, hepatoma, and retinalgt11 cDNA libraries were plated at densities of ˜10,000 plaques/150-mmPetri dish. Initially, the placental library was screened using a 404-bpBstEII fragment isolated from the type 2 human ASM partial cDNA, pASM-2(Quintern et al., 1989, EMBO J. 8:2469-2473). This fragment containedthe type 2-specific 40-bp region, as well as flanking sequences commonto the type 1 and 2 cDNAs. The hepatoma cDNA library was screened withan oligonucleotide (5′-GTTCCTTCTTCAGCCCG-3′) (SEQ ID NO: 24) constructedfrom the 5′ end of the longest partial type 1 cDNA previously isolatedand then was analyzed for the presence of type 2 cDNAs as describedbelow. The testis library was screened first with the type 2-specificoligonucleotide (40-mer) and then with a 608-bp PstI-SacI restrictionfragment isolated from a type 2 cDNA (Quintern et al., supra). Theretinal library was screened with the full-length type 1 placental cDNA(PASM-1FL). Random primer labeling of the cDNA probe with [γ-32P]dCTP(˜3000 Ci/mmol), 5′ end labeling of the PstI oligonucleotides with T4polynucleotide kinase and [γ-32P]ATP (>5000 Ci/mmol), and filterhybridizations were performed by standard methods (Sambrook et al.,1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.). After three rounds ofpurification. DNA was isolated from putative positive plaques by theplate lysate method, and the cDNA inserts were analyzed on 1% agarosegels by Southern hybridization (Southern, 1975, J. Mol. Biol.98:503-517) with oligonucleotides constructed from the type 1- and2-specific regions and ASM intronic sequences.

6.1.4. DNA Sequencing and Computer-Assisted Analyses

Dideoxy sequencing was performed by the method of Sanger et al. (Sangeret al., 1977, Proc. Natl. Acad. Sci. U.S.A. 74:5463-5467). A putativepositive full-length human type 1 ASM cDNA insert (pASM-1FL,) and arepresentative partial type 3 cDNA insert (pASM-3) were isolated afterdigestion with EcoR1 and electrophoresis through agarose gels. Briefly,the inserts were cut out of the gels, phenol was added, and then thesamples were freeze-thawed in an ethanol bath. Following centrifugationat ˜10,000×g, the aqueous phases were re-extracted with phenol andphenol:chloroform (1:1, v/v), and the DNA was then isolated by ethanolprecipitation. The purified inserts were subcloned in both orientationsinto the Bluescript vector SK(+) for sense and antisense strands, andsingle-stranded template was rescued using VCS13 helper phage fordideoxy sequencing according to the manufacturer's instructions.Sequencing primers were synthesized on an Applied Biosystems model 380BDNA synthesizer using phosphoramidite chemistry (Itakura et al., 1984,Annu. Rev. Biochem. 53:323-356). Computer analyses were performed usingthe University of Wisconsin Genetics Computer Group DNA sequenceanalysis software (version 6.2) and GenBank (release 64) and NBRF(release 25) DNA and protein bases, respectively.

6.1.5. Analysis of Polymorphic-Sites in the Human ASM Coding Region

To determine the population frequency of the base differences in codons322 and 506 of the full-length ASM transcript, PCR amplification ofgenomic DNA from 20 normal Caucasian individuals was performed on aPerkin Elmer-Cetus thermal cycler using Taq polymerase according to themethod of Saiki et al. (Saiki et al., 1988, Science, 239:487-491). Forthe codon 322 base difference, sense (5′-AGTAGTCGACTGCTAGAGCAATCAGAG-3′)(SEQ ID NO: 25) and antisense (5′-AGTGTCGACTCGTCAGGACCAAC-3′) (SEQ IDNO: 26) PCR primers were constructed as described above to amplify a375-bp genomic DNA fragment. For the codon 506 base difference, sense(5′-AGTAGTCGACATGGGCAGGATGTGTGG-3′) (SEQ ID NO: 17) and antisense(5′-AGTAGTGTCGACTTGCCTGGTTGAACCACAGC-3′) (SEQ ID NO: 16) primers wereconstructed to amplify a 567-bp genomic DNA fragment. For these studiesgenomic DNA was rapidly isolated from whole blood by the followingprocedure. About 0.5 ml of whole blood and 0.5 ml of lysis buffer (10 mMTris/HCl buffer, pH 7.5, containing 5 mM MgCl₂, 0.32 M sucrose, and 1%Triton X-100) was mixed at room temperature. Following centrifugation at13,000×g, the supernatant was removed, and 0.5 ml of PCR buffer (10 mMTris/HCl buffer, pH 8.3, containing 50 mM KCl, 2.5 mM MgCl₂, 0.1 mg/mlgelatin, 0.45% Nonidet P-40, 0.45% Tween 20, and 0.1 mg/ml Proteinase K)was added. The samples were incubated at 60° C. for 1 h and boiled for10 minutes to inactivate the protease, and then 25 μl was removed forPCR amplification. Following agarose gel electrophoresis of the PCRproducts, the concentration of each product was estimated by ethidiumbromide staining.

For the dot-blot hybridization analyses (Sambrook et al., supra), about0.5 μg of DNA was used. The sequence-specific oligonucleotide probeswere 5′-ATGAAGCAATACCTGTC-3′ (Ile-322) (SEQ ID NO: 27),5′-ATGAAGCAACACCTGTC-3′ (Thr-322) (SEQ ID NO: 28),5′ACTACTCCAGGAGCTCT-3′(Arg-506) (SEQ ID NO: 29), and5′-ACTACTCCGGGAGCTCT-3′ (Gly-506) (SEQ ID NO: 30). Hybridizations wereperformed for at least 3 h at 42° C. Following hybridization, the blotswere washed at room temperature for 15 minutes in 6×SSC containing 0.1%sodium dodecyl sulfate and then for 2 h in the same solutions at either51° C. (Ile-322 and Arg-506) or 53° C. (Thr-322 and Gly-506).

6.1.6. Reconstruction of Full-Length Type 2 and 3 cDNAS

Since extensive library screening did not identify full-length type 2 or3 cDNAs, they were reconstructed as outlined in FIGS. 5, A and B. Forthe full-length type 2 cDNA (pASM-2FL) a 400-bp BstEII restrictionfragment containing the type 2-specific 40-bp sequence was isolated fromthe partial type 2 cDNA, pASM-2, by the phenol/freeze-thaw methoddescribed above. The full-length type 1 cDNA, pASM-1FL, was thendigested with BstEII to remove the fragment containing the type1-specific sequence for replacement with the type 2-specific BstEIIfragment. Analogously, the full-length type 3 cDNA (pASM-3FL) wasreconstructed using a 360-bp type 3-specific BstEII restrictionfragment. (FIG. 5B).

6.1.7. Transient Expression in COS-1 Cells and Stable Expression in CHOCells

For transient expression experiments in COS-1 cells, the full-lengthpASM-1FL insert and the reconstructed full-length pASM-2FL and pASM-3FLcDNA inserts were subcloned in both orientations into the EcoRI site ofthe eukaryotic expression vector, p91023(B) (Kaufman and Sharp, 1982,Mol. Cell. Biol. 2:1304-1319). Full length pASM-1FL inserted inexpression vector, p91023(B) was designated p91-ASM. DNA (˜5-20 μg) fromsense and antisense constructs was then transfected into COS-1 cells bythe method of Chen and Okayama (Chen and Okayama, 1987, Mol. Cell. Biol.7:2747-2752). The transfected cells were harvested after 72 h, and theASM and β-glucuronidase enzymatic activities were determined usingN-12[(12 pyrenesulfonyl)amidododecanoyl]-sphingomyelin and4-methylumbelliferyl-β-glucuronide, respectively (Klar et al., 1988,Clin. Chem. Acta 176:259-268; Brot, et al., 1978, Biochemistry17:385-391). Neutral sphingomyelinase activities were determined aspreviously described (Gatt et al., 1978, J. Neurochem. 31:547-551). Aunit of enzymatic activity equaled that amount of enzyme which hydrolyed1 nmol of substrate/h. Protein determinations were performed by amodified fluorescamine assay (Bishop and Desnick, 1981, J. Biol. Chem.256:1307-1316). In addition to antisense constructs, mock transfectionswere performed as controls.

For stable expression the CHO DG44 dhfr⁻ cell line was utilized (Urlauget al., 1986, Somat. Cell Genet. 12: 555-566). The CHO cells weremaintained at 37° C. in 5% CO₂ in Dulbecco's Modified Eagle Medium(DMEM) with 10% fetal calf serum (FCS), antibiotics, 0.05 mMhypoxanthine and 0.008 mM thymidine. Following transfection, therecombinant CHO lines were grown in DMEM supplemented with 10% dialyzedFCS in the absence or presence of methotrexate (MTX).

6.1.8. PCR Amplification of Genomic DNA

For the genomic PCR reactions, sense and antisense primers weresynthesized as described above. PCR primers 1 and 2 were constructedfrom exonic sequences which were common to type 1 and 2 cDNAs (see FIGS.6A-6B). Genomic DNA was isolated from cultured normal fibroblasts bystandard methods (Sambrook et al., supra). PCR amplifications wereperformed for 30 cycles, and the amplified products were analyzed asdescribed above. For DNA sequencing, the fragments were isolated fromthe agarose gels, subcloned into Bluescript vectors, and sequenced bythe methods described above.

6.1.9. The Genomic Structure of ASM

A human genomic library (average insert size˜10-15 kb) was constructedin the phage vector EMBL 3 (Promega) and kindly provided by Dr. RuthKornreich, Mount Sinai School of Medicine, NY. This library was screenedat a density of ˜10,000 plaques/150 mm petri dish using the full-lengthtype 1 ASM cDNA, pASM-1FL (Schuchman et al., in review). Filtertransfers and Southern hybridizations were performed by standard methods(Sambrook et al. 1989). Random primed labeling of the cDNA probe wasperformed using [α-³²P] CTP (˜3000 Ci/mmol) (Amersham) according to themanufacturers instructions. Following three rounds of plaquepurification, DNA was isolated from positive clones by the plate lysatemethod (Sambrook et al. 1989) and analyzed by Southern hybridizationwith oligonucleotides (17 mers) spanning the entire coding region of thefull-length ASM cDNA. 5′ end labeling of the oligonucleotides wasperformed with T4 polynucleotide kinase (New England Biolabs) and[γ-³²P] ATP (>5000 Ci/mmol) (Amersham). Oligonucleotide hybridizationswere performed by standard methods (Sambrook et al. 1989).

Dideoxy sequencing was performed by the method of Sanger et al. usingSequenase DNA sequencing kits (United States Biochemical). An ˜8 kb SalI/Eco RI restriction fragment which contained the entire ASM codingregion was isolated from an ASM genomic clone, ASMg-1, and digested withHinc II (Promega) to generate four fragments of about 2.8, 2.0, 1.6 kband 1.4 kb, respectively. The genomic restriction fragments weresubcloned into the Bluescript vector, SK(+) (Stratagene), and sequencedin both orientations by double-stranded sequencing methods. Sequencingprimers were synthesized on an Applied Biosystems DNA Synthesizer usingphosphoramidite chemistry (Itakura et al. 1984). Computer analyses wereperformed using the University of Wisconsin Genetics Computer Group DNASequence Analysis Software (version 6.2) and GenBank (release 64) andNBRF (release 25) DNA and protein databases, respectively. The resultsof this analysis are depicted in FIGS. 6C-6H.

6.2. Results 6.2.1. Evidence for the Occurrence of Type 1 and 2 ASMTranscripts

Since only partial type 1 and 2 ASM cDNAs had been previously isolatedfrom human placental and fibroblast cDNA libraries (Quintern et al.,supra), Northern hybridization analyses were performed to determine therespective sizes and relative amounts of the type 1 and 2 transcripts.As shown in FIG. 1A, a single band of ˜2.5 kb was detected when poly(A)⁺RNA from human placenta was hybridized with the partial type 1 cDNA(which could detect both type 1 and 2 transcripts). Longer exposures ofup to 7 days did not reveal additional hybridizing bands (data notshown). Therefore, to demonstrate the occurrence of type 1 and 2 ASMtranscripts, RNase protection experiments were carried out. Asillustrated in the schematic (FIG. 1B, right), using a type 1radiolabeled riboprobe (see “Methods”), it was expected that a type 1transcript would have a 333-bp protected fragment, while a 266-bpfragment would be protected in a type 2 transcript. In human placentalpoly(A)⁺ RNA, both type 1- and 2-specific transcripts were detected(FIG. 2B). Lanes 2 and 3 show 16- and 40-h exposures, respectively,while lane 1 was a control protection assay performed without theaddition of poly(A)⁺ RNA. Together with the Northern hybridizationresults, these experiments indicated that human placenta contained type1 and 2 transcripts of ˜2.5 kb. Furthermore, densitometric quantitationrevealed that the type 2 transcripts represented from 5 to 10% of thetotal ASM placental RNAs, consistent with the previous cDNA libraryscreening results.

To further demonstrate the occurrence of full-length type 2 cDNAs, PCRamplification experiments were performed using a sense primer from the5′ end of the full-length type 1 cDNA (pASM-1FL; see below) and anantisense primer constructed from the type 2-specific 40-bp region (FIG.2C). As shown in FIG. 2A, a product of the expected size (˜1.1 kb) wasamplified, which specifically hybridized with an ASM-specificoligonucleotide probe (FIG. 2B). Control experiments also were performedusing these primers to PCR-amplify the full-length type 1 cDNA (data notshown). As expected, no amplified products were found, demonstrating thespecificity of this PCR for type 2-containing sequences. DNA sequencingof the amplified ˜1.1-kb cDNA fragment revealed that the type 1 and 2cDNAs had identical 5′ sequences.

6.2.2. Isolation and Characterization of a Full-Length Human Type 1 ASMcDNA

Since the longest type 1 and 2 ASM cDNAs previously isolated were 1879and 1382 bp, respectively (Quintern et al., supra), intensive cDNAlibrary screenings were undertaken to isolate the respective full-lengthASM cDNAs. Screening of ˜2×10⁶ independent recombinants from a humanplacental cDNA library resulted in the isolation of 84 putative positivehuman ASM cDNA clones. Agarose gel electrophoresis and Southernhybridization analyses revealed that the cDNA inserts ranged from ˜1.2to 2.4 kb and that ˜90% were type 1. Of the nine type 2 cDNAs isolated,the longest insert was ˜1.4 kb.

Clone pASM-1FL, isolated from the placental library, contained thelongest type 1 insert, a 2347-bp sequence which included an 87-bp5′-untranslated region, an 1890-bp open reading frame encoding 629 aminoacids, and a 370-bp 3′-untranslated region (FIGS. 3A-3C) (SEQ ID NO: 1).The coding region contained six N-glycosylation consensus sequences(encoding Asn-X-Thr/Ser) at residues 86-88, 175-177, 335-337, 395-397,503-505, and 522-524. Although no poly(A)⁺ tail was present, a consensuspolyadenylation sequence was found at nucleotides 2254-2259, consistentwith its position in pASM-1 and pASM-2 (Quintern, et al., supra). Therewere two in-frame ATGs present in the 5′ region of the pASM-1FL insert,beginning at nucleotides 1 and 97. Using the von Heijne weight-matrixmethod (von Heijne, 1986, Nucleic Acids Res. 14:4683-4690), the signalpeptidase cleavage site was optimally predicted after residue 46 (vonHeijne score=10.8; FIG. 4, ). The next best signal peptide cleavage sitewas after residue 50 (von Heijne score=10.1). Interestingly, thepredicted signal peptide consisted of a hydrophobic core sequence, whichcontained five repeats of the amino acid residues leucine and alanine;the corresponding nucleotide sequence contained a 12-nt tandem directrepeat at nucleotides 109-133.

The predicted amino acid sequence of the pASM-1FL insert was colinearwith 111 microsequenced residues in tryptic peptides of ASM purifiedfrom human urine (Quintern et al., 1987, Biochim. Biophys. Acta922:323-336). The four discrepancies between the predicted pASM-1FLamino acid sequence and the microsequenced peptides also occurred in thesequences predicted by the pASM-1 and pASM-2 inserts (Quintern, et al.,1989, supra). However, comparison of the predicted amino acid sequencesof the full-length type 1 placental cDNA (pASM-1FL) and the previouslyreported type 1 (and 2) fibroblast cDNAs revealed two other differences.In the placental cDNA, codons 322 (ATA) and 506 (AGG) predictedisoleucine and arginine residues, respectively, whereas in thefibroblast cDNAs the predicted amino acids were threonine (322) andglycine (506) due to single base changes (ACA and GGG, respectively).Dot-blot hybridization studies of 20 normal Caucasian individuals withsequence-specific oligonucleotide probes (data not shown) revealed thatthe Gly-506 codon had an allele frequency of 0.7 and the Thr-322 codonhas an allele frequency of 0.6, indicating that these nucleotidedifferences were common polymorphisms.

6.2.3. Isolation and Characterization of a Type 3 Human ASM cDNA

Since no full-length type 2 cDNAs were isolated from the placentallibrary, efforts were directed to screen testis, retinal, and hepatomacDNA libraries. Screening of ˜2×10⁶ independent recombinants from ahuman testis library with a type 2-specific 40-mer did not detect anytype 2 cDNAs. Replica filters were then screened with the type 2 cDNA,pASM-2. Again, no type 2 cDNAs were identified, although 93 type 1clones were isolated and analyzed. Next, a human retinal cDNA librarywas intensively screened with pASM-1FL. From ˜5×10⁶ independentrecombinants screened, 26 putative positive ASM cDNAs were isolated andanalyzed by Southern hybridization. Of these, there were 10 type 1 andone type 2 cDNAs. Again, only partial type 2 cDNAs were identified. Theremaining cDNAS isolated from this library were too short to determineif they were type 1 or 2. Finally, ˜1.0×10⁶ recombinants in the hepatomalibrary were screened with an oligonucleotide constructed from the 5′end of pASM-1, the longest partial type 1 cDNA previously isolated. Fiveputative full-length ASM cDNAs were isolated; however, Southernhybridization analysis demonstrated that they had all type 1 cDNAinserts. Notably, restriction enzyme analysis of the 65 partial humanASM cDNAs isolated from the testis library revealed a third type ofhuman ASM cDNA (pASM-3, type 3). As shown schematically in FIG. 4, thepASM-3 cDNA was 1914 bp and did not contain either the type 1-specific172-bp region or the type 2-specific 40-bp sequences, but had atruncated open reading frame of 934 bp.

6.2.4. Reconstruction of Full-Length Type 2 and 3 cDNAs and TransientExpression of the Full-Length ASM cDNAs

Since intensive screening of five different cDNA libraries did notidentify full-length type 2 or 3 cDNAs, full-length sequences werereconstructed by the procedure shown in FIGS. 5A-5C to test theirfunctional integrity. The reconstructions were based on the fact thatPCR amplification and DNA sequencing studies (shown in FIG. 2 for thetype 2 cDNA) had revealed that the full-length type 2 and 3 sequencesexisted in human placenta and that the 5′ sequences were identical tothat found in the full-length type 1 insert, PASM-1FL.

The pASM-1FL insert and the reconstructed full-length type 2 (pASM-2FL)and 3 (pASM-3FL) cDNAs were inserted into the transient expressionvector, p-91023(B) (Kaufman et al., supra) and transfected into COS-1cells. As shown in Table I, the mean endogenous ASM activity in COS-1cells toward fluorescent natural substrate,N-12[(1-pyrenesulfonyl)amidododecanoyl]-sphingomyelinase was about 7.1units/mg protein. The ASM activity in COS-1 cells transfected with theantisense constructs ranged from about 6.2 to 6.7 units/mg protein. Incontrast, COS-1 cells transfected with the p-91023(B) full-length type 1sense construct had 30.6 units/mg protein of ASM activity (˜5-fold overendogenous levels), demonstrating that the pASM-1FL type 1 transcriptexpressed catalytically active enzyme. The reconstructed type 2 and 3transcripts did not express catalytically active enzymes in COS-1 cells.None of the ASM full-length transcripts expressed neutralsphingomyelinase activities in COS-1 cells. As an additional control,the activity of another lysosomal enzyme, β-glucuronidase, also wasdetermined and did not vary significantly from the endogenous levels inany of the transfection experiments.

TABLE I Transient Experiments of Human Acid Sphingomyelinase inCOS-Cells Values represent the average of two independentdeterminations. Source Acid sphingomyelinase β-Glucuronidase nmol/h/mgCOS-1 cells 7.1 283 Type 1 Sense 30.6 314 Antisense 6.7 338 Type 2 Sense6.7 307 Antisense 6.2 342 Type 3 Sense 7.4 288 Antisense 6.4 261

6.2.5. PCR Amplification of ASM Genomic DNA

In order to determine the origin of the type 1, 2, and 3 cDNAs, an ASMgenomic region was PCR-amplified with primers constructed from commonexonic sequences flanking the type 1- and 2-specific sequences (FIGS.6A-6B). A 1665-bp PCR product was isolated and sequenced. This genomicregion contained both the 172- and 40-bp type 1- and 2-specificsequences. Interestingly, the 172-bp type 1 sequence was exonic, flankedby 1052-bp and 229-bp introns. The 40-bp type 2-specific sequence waslocated at the 5′ end of the 1052-bp intron. Within this intron therealso were two poly(T) tracts of 20 and 23 nt at positions 313-332 and469-491 and five potential lariat branch points (labeled a-e, FIG. 6).Within the 229-bp intron there are two potential lariat branch pointsthat fit the consensus sequence, YNYURAY (Padgett et al., 1986, Annu.Rev. Biochem. 55: 1119-1150; Green, 1986, Annu. Rev. Genet. 20:671-708),at positions 1574-1580 and 1510-1516 (underlined in FIG. 6). The secondpotential branch point, 77 nt upstream from the A2 acceptor splice site,is followed by a polypyrimidine tract.

Table II shows the donor (D1 and D2) and acceptor (A1 and A2) splicesite sequences at the intron/exon boundaries within this ASM genomicregion, as well as the sequence of the cryptic donor splice site (D3)located adjacent to the 3′ end of the 40-bp type 2-specific sequence.Note that neither of the donor sites within this region perfectlymatched the consensus sequence (von Heijne, supra) and, in particular,there was a G to A transition within donor splice site D2, located atthe 3′ end of the type 1-specific.172-bp exon. Compared to the D1 and D2donor sites, the D3 cryptic splice site adjacent to the 40-bp type2-specific sequence best matches the donor consensus sequence.

TABLE II 5′ Donor and 3′ Acceptor Splice Sites in the PCR-AmplifiedGenomic ASM Region Splice site Nucleotide sequence position DonorAcceptor Consensus CAG gtaagt ncag G A g T D1 136-144 CAG gta{dot over(ct)}t D2 1360-1368 AA{dot over (A)} gtgag{dot over (g)} D3 176-184 aaggtga{dot over (a)}t A1 1187-1190 tcag {dot over (A)} A2 1588-1592 c{dotover (t)}ag G

6.2.6. Stable Expression in CHO Cells

Recombinant clones stably expressing human ASM were obtained byelectrotransfection of the p91-ASM construct into DG44 dhfr⁻ CHO cellsand amplification of the integrated vector DNA with selection inincreasing MTX concentrations. Initial growth in media lackingnucleosides resulted in the identification of over 20 clones expressingASM at levels ranging from 5-30 units/mg. Clones with the highest ASMlevel were grown in the presence of 0.02 to 0.32 μM MTX to amplify theintegrated p91-ASM DNA. The MTX induced amplification at 0.32 μM MTXresulted in the intracellular production of ASM at levels of over 300units/mg. Importantly, human ASM was secreted into the media at levelsat least five times over endogenous.

6.2.7. Engineering of Transgenic Mice Containing the Human ASM Gene

One of the most important goals of modern molecular biology is tounderstand how mammalian genes are regulated. Although in vitroexpression systems have provided valuable insights into the mechanismsunderlying mammalian gene regulation, to fully decipher the complexarray of developmental and tissue-specific regulatory mechanismsoperating in mammals, in vivo expression systems must be utilized.Clearly, one of the most powerful systems for the in vivo analysis ofmammalian gene regulation is the use of transgenic mice. For example, wehave been investigating the gene encoding the human lysosomal enzymeASM. In order to study the regulation of this gene, a 12 kilobase (kb)ASM genomic fragment was isolated which included the complete codingregion of the ASM polypeptide and about 4 kb of upstream sequences. Thispurified genomic fragment was given to the Transgenic Mouse CoreFacility, where it was microinjected into about 30 mouse male pronuclei.Following microinjection, the pronuclei were implanted intopseudopregnant females and allowed to develop in vivo. Two founderanimals were produced which contained the integrated human ASM gene.Both founder animals transmitted the human gene to their offspring,demonstrating that their germ cells contained the human ASM sequences.Interestingly, offspring from both founder animals expressed high levelsof human ASM activity, suggesting that the sequences required for ASMtranscriptional activity are contained within the 12 kb genomicfragment.

6.3. Discussion

Previously, we reported the isolation of partial type 1 and 2 cDNAs forhuman ASM, the longest inserts being 1879 (pASM-1) and 1382 (pASM-2) bp,respectively. Type 1 cDNAs had a unique 172-bp sequence encoding 57amino acids which was replaced in the type 2 cDNAs by a 40-bp sequenceencoding 13 different amino acids. About 90% of the 113 partial cDNAsisolated from human fibroblast and placental libraries were type 1. Inthe studies reported here, Northern hybridization analyses revealed thepresence of a single ˜2.5 kb transcript in placental poly(A)⁺ RNA.Subsequent RNase protection studies demonstrated the occurrence of bothtype 1 and 2 transcripts. Thus, efforts were undertaken to isolatefull-length type 1 and 2 ASM cDNAs by intensive screening of cDNAlibraries from five different tissues.

Full-length type 1 cDNAs were isolated including the 2347-bp cDNA,pASM-1FL. The size of the pASM-1FL insert was consistent with theoccurrence of the ˜2.5 kb transcript observed in the Northernhybridization experiments, the 150-bp difference due to upstream5′-untranslated sequences, and the length of the poly(A) tract. Thefull-length cDNA had an open reading frame of 1890 bp which containedtwo in-frame potential initiation codons. Since the enzyme's N terminuswas blocked (Quintern et al., 1989, EMBO J 8:2469-2473), it is not knownwhich initiation codon was used in vivo. It is possible that bothinitiation ATGs could be used as is the case for another hydrophobiclysosomal hydrolase, acid β-glucosidase (Sorge et al., 1987, Proc. Natl.Acad. Sci. U.S.A. 84:906-910). However, compared to the translationinitiation consensus sequence GCC(AG)CCATGG (Kozak, 1987, Nucleic AcidsRes. 15:8126-8149), the sequence flanking the second ATG in human ASM isweak, particularly since position −3 contains a thymidine residue.Therefore, it is likely that the first ATG is the in vivo initiationcodon. Using the von Heijne weight-matrix method (von Heijne, 1986,Nucleic Acids. Res. 14:4683-4690), the optimal signal peptide cleavagewould occur after amino acid residue 46. The predicted 14 amino acidspreceding the signal peptide cleavage site have a particularlyhydrophobic core consisting of five leucine/alanine repeats. Sixpotential N-glycosylation sites were predicted in the mature ASMpolypeptide. At present it is not known which of the glycosylation sitesare utilized. However, treatment of the purified urinary enzyme withglycopeptidase F reduced the molecular mass from −72 kDa to −61 kDa,indicating that perhaps as many as five sites may be glycosylated(Quintern, et al., supra).

Efforts to identify a full-length type 2 ASM cDNA included intensivescreening of five libraries. Screening of placental and retinallibraries resulted in the identification of fourteen additional type 2cDNAs ( )12% of the total cDNAs analyzed); however, none were longerthan the previously obtained pASM-2 insert (1382 bp). Screening ofhepatoma and testis libraries did not identify any type 2 clones, but athird 1914-bp ASM cDNA (type 3, pASM-3) was identified which lacked boththe unique type 1 and 2 sequences.

In order to determine the functional integrity of the three differentASM transcripts, it was necessary to reconstruct full-length type 2 and3 coding sequences (FIG. 5). Prior to performing these reconstructionexperiments, the existence of full-length type 2 and 3 cDNAs was shownby PCR amplification of total placental cDNA (shown in FIG. 2 for thetype 2 cDNA) and sequencing of the amplified products. Transientexpression in COS-1 cells of pASM-1FL and the reconstructed type 2 and 3cDNAs demonstrated that only the type 1 transcript was functional. Thefact that the type 1 mRNA did not express neutral sphingomyelinaseactivity in COS-1 cells supports the notion that the acid and neutralsphingomyelinases are encoded by different genes.

Two nucleotide differences were initially identified by sequencing type1 cDNAs from fibroblast and placental libraries (Quintern et al.,supra). The functional integrity of the full-length pASM-1FL sequencefrom placenta (i.e. Ile-322 and Arg-506) was demonstrated by thetransient expression of active enzyme in COS-1 cells (Table I).Subsequent analysis of genomic DNA from 20 unrelated individualsrevealed that the base differences in codons 322 and 506 occurred in theCaucasian population as polymorphisms with allele frequencies of 0.6 and0.7 for the Thr-322 and Gly-506 codons, respectively. The Gly-506polymorphism creates MspI and NciI restriction sites.

The origin of the type 1, 2 and 3 transcripts was deduced by analysis ofthe PCR amplified genomic region, which included both the unique type 1and 2 sequences (FIGS. 6A-6B) (SEQ ID NO: 3). The 172-bp type 1-specificsequence was an exon, flanked by introns of 1052 and 229 bp, whereas the40-bp type 2-specific sequence was intronic, having been derived fromthe 5′ end of the 1052-bp intron. No ASM transcripts were found whichcontained both the unique type 1- and 2-specific sequences. Thesefindings are consistent with alternative splicing of a single ASM hnRNA.As shown diagrammatically in FIG. 7, type 1 transcripts result fromnormal splicing events which remove the 1052- and 229-bp introns, whiletype 2 transcripts result from splicing at a cryptic site which excises1012 bp of the large intron, the 172-bp type 1 exon, and the 229-bpintron. The occurrence of the type 3 cDNA can be explained byalternative splicing to the upstream donor splice site, D1, therebydeleting the 172-bp exon.

Splicing of mammalian transcripts is initiated by cleavage at the 5′donor splice site followed by lariat formation at a branch point,generally within 50 nt of the 3′ acceptor site (Padgett et al., 1986,Annu. Rev. Biochem. 55:1119-1150; Green, 1986, Annu. Rev. Genet.20:671-708). Then there is a cleavage of the 3′ exon at the acceptorsplice site and ligation of the adjacent exons. However, in the humanASM mRNA, there is a weak 5′ donor splice site adjacent to the 172-bpexon (D2, Table II) that does not function in about 10% of the splicingevents, thereby generating the type 2 or 3 transcripts. As shown in FIG.7, if the cryptic donor splice site (D3) adjacent to the 40-bp intronicsequence is used, a type 2 transcript is generated. The G to Atransition of the nucleotide immediately adjacent to the invariant GTconsensus dinucleotide in D2 may be particularly important, since thisalteration has been shown to cause abnormal splicing of the proα1(I)collagen gene leading to Ehlers-Danlos syndrome Type VII (Weil, et al.,1989, EMBO J 8:1705-1710). In fact, the D3 cryptic splice site moreclosely matches the consensus sequence than either of the other twoidentified donor splice sites, D1 or D2, which are used to generate type1 transcripts (Table II). The presence of two poly(T) tracts of 20 and23 nt at the 5′ end of the large intron may cause abnormal RNA secondarystructure, perhaps favorably positioning the cryptic splice site, D3.The rare type 3 transcript also is generated by alternative splicing ofthe 172-bp exon, but in this case splicing proceeds to the upstreamdonor splice site D1, rather than to the D3 cryptic donor splice site.

Other features of the 1052-bp intron also deserve note. There are fivepotential lariat branch point sequences that fit the consensus sequenceYNYURAY (Padgett, et al., supra) located near the 3′ end of this intron(labeled a-e in FIG. 6A). Only one of these potential branch points isfollowed by a polypyrimidine tract (b), however this branch point is 336bp upstream from the 3′ acceptor site. It is generally assumed that thebranch point should be within 20-50 nt of the 3′ acceptor and not closerthan 70 nt to the 5′ donor site. Perhaps after cleavage at the D3cryptic donor splice site, a lariat cannot efficiently form at branchpoints within this intron, and thus, the lariat occurs at the nextavailable branch point, which is located in the 229-bp intron.

Alternative splicing also occurs in the transcripts for two other humanlysosomal enzymes, β-glucuronidase (Oshima et al., 1987, Proc. Natl.Acad. Sci. U.S.A. 84:685-689) and β-galactosidase (Morreau et al., 1989,J. Biol. Chem. 264:20655-20663). β-Glucuronidase cDNAs, which had aninternal 153-bp deletion, were identified in human fibroblast andplacental cDNA libraries. The shorter cDNA had lost an entire exon dueto alternative splicing and expressed an enzyme protein that was notcatalytically active in COS-1 cells. For β-galactosidase, two distinctgroups of cDNA clones were isolated from human fibroblast cDNAlibraries. The shorter cDNAs were missing two noncontiguous proteincoding regions present in the full-length cDNAs and produced truncatedproteins, which were targeted to the perinculear region in COS-1 cells.

In summary, three types of human ASM transcripts have been identified.Genomic PCR amplification and sequencing studies demonstrated that eachof the ASM transcripts were derived from a single mRNA. The type 1transcript is the major ASM species and encodes a catalytically activeenzyme. The type 2 and 3 ASM transcripts result from alternativesplicing, most likely due to the presence of a weak donor splice site(D2) adjacent to the 172-bp type 1-specific exonic sequence.Reconstruction and transient expression of full-length type 2 and 3transcripts revealed that these sequences did not encode functionalenzymes. The availability of the full-length type 1 cDNA will permitcharacterization of the human ASM gene and structure/function studies ofthe ASM polypeptide, as well as investigations of the mutations whichcause the neuronopathic and non-neuronopathic forms of Niemann-Pickdisease.

7. Example: Niemann-Pick Disease: a Frequent Missense Mutation in theAcid sphingomyelinase-Encoding Gene of Ashkenazi Jewish Type A and BPatients 7.1. Materials and Methods 7.1.1. Cell Lines

Primary cultures of fibroblasts and lymphoblasts were established fromskin biopsies and peripheral blood samples obtained from NPD patientsand family members and from normal individuals; informed consent wasobtained. NPD cell lines GM00112A, GM00165, GM00370, GM00406, GM00559,GM02895, and GM03252 were obtained from the National Institute ofGeneral Medical Sciences Human Genetic Mutant Cell Repository Institutefor Medical Research (Camden, N.J.). Cell lines 444X.F01, 534R.F03,556X.F01, 888V.F01, 2789X.F01, 4293Q.E02, 4774Z.F01, 5113C.L01,5115E.F01, and 6791M.F01 were obtained from the Service de Biochimie,Hospice de Lyon (Lyon, France). Cell lines DMN 83.126, DMN 84.135, DMN86.40, DMN 86.49, DMN 87.71, DMN 87.99, DMN 88.9, DMN 83.133, GJO, andRNS were provided by Peter Penchev (Developmental and MetabolicNeurology Branch, National Institute of Neurological and CommunicativeDisorders and Stroke). The cells were grown in RPMI 1640 medium/10%fetal bovine serum/1% penicillin/streptomycin at 1 mg/ml by standardprocedures (Bernstein, et al., 1989, J. Clin. Invest. 83:1390-1399). Thediagnosis of types A and B NPD was based on clinical criteria (e.g., ageat onset, presence of neurologic involvement, etc.) and by demonstrationof markedly deficient ASM activity in cultured cells (Klar et al., 1988,Clin. Chim. Acta 176:259-268).

7.1.2. Enzyme and Protein Assays

ASM activity was determined in cultured fibroblasts obtained from NPDpatients and normal individuals using the fluorescent natural substrate.[N-12(1-pyrenesulfonyl)amino dodecanoyl]sphingomyelin as described(Klar, et al., supra). One unit of activity equals that amount of enzymethat hydrolyzes 1 nmol of substrate per hr. Protein was determined by amodified fluorescamine assay (Bishop and Desnick, 1981, J. Biol. Chem.256:1307-1316).

7.1.3. cDNA and Genomic Amplification and Sequencing of the MutantAllele

Total RNA and genomic DNA were isolated from cultured skin fibroblastsby standard procedures (Sambrook et al., 1989, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.). First-strand cDNA was reverse-transcribed from )5 μg of total RNAby using a cDNA synthesis kit according to the manufacturer'sinstructions (Boehringer Mannheim). The cDNA ( )10% of the totalreaction) or genomic DNA ( )0.5 μg) was amplified by the PCR withThermus acquaticus (Taq) polymerase (Perkin-Elmer Cetus) using aPerkin-Elmer Cetus thermalcycler, essentially as described by Saiki etal. (Saiki et al., 1988, Science 239:487-491) with the followingconditions and modifications. PCR was performed for 30 to 40 cycles andconsisted of denaturation for 1 minute at 94° C. and hybridizing andextension for 4 minutes at 66° C. To improve the specificity of the PCRamplification, a “PCR boost” procedure was used. In this procedure theconcentrations of the primers and Taq polymerase were 0.1 μM and 5units/ml, respectively, for the first 15 cycles. Then each primer wasadded to a final concentration of 0.5 μM, and an additional 2 units ofTaq polymerase was added to the reaction mixture. PCR amplification thenproceeded for an additional 15-25 cycles.

Pairs of sense and antisense oligonucleotide primers were synthesized onan Applied Biosystems model 380B DNA synthesizer (Itakura, et al., 1984,Annu. Rev. Biochem. 53:323-356) and used to specifically amplify (i) theentire coding region of the reverse-transcribed type 1 ASM transcript inthree overlapping cDNA fragments, (ii) the 1665-bp genomic regioncontaining the alternatively spliced sequences in the type 1 and 2 ASMcDNAS (E.H.S., unpublished work) and (iii) a genomic region thatincluded the point mutation for confirmation of the candidate missensemutation. To amplify a 984-bp fragment from the 5′ end of the ASM cDNA,the 29-mer sense primer, P1(5′-AGTAGTCTCGAGACGGGACAGACGAACCA-3′) (SEQ IDNO: 11) corresponded to ASM nucleotide (nt) −39 to −23 with anadditional 12 nt that included an XhoI restriction site and the 31-merantisense primer. P2 (5′-AGTAGTCTGCAGAGCAGGGTACATGGCACTG-3′) (SEQ ID NO:12) corresponded to ASM nt 926 to 945 with an additional 12 ntcontaining an EcoRI restriction site. To amplify an internal 383-bpfragment of the ASM cDNA, the 29-mer sense primer, P3(5′-ATCATCAAGCTTGGGTAACCATGAAAGCA-3′) (SEQ ID NO: 13), corresponded toASM nt 947-964 with an additional 12 nt containing a HindIII restrictionsite, and the antisense 32-mer primer. P4(5′-ATCATCGAATTCTACAATTCGGTAATAATTCC-3′) (SEQ ID NO: 14), correspondedto ASM nt 1310 to 1330 with an additional 12 nt containing an EcoRIrestriction site. To amplify a 789-bp 3′ fragment from the ASM cDNA, a19-mer sense primer, P5 (5′-CTCCACGGATCCCGCAGGA-3′) (SEQ ID NO: 15),corresponded to ASM nt 1855 to 1203 and contained an internal BamHIrestriction site, and an antisense 32-mer primer. P6(5′-AGTAGTGTCGACTTGCCTGGTTGAACCACAGC) (SEQ ID NO: 16), corresponded toASM nt 1955 to 1974 with an additional 12 nt containing a SalIrestriction site. Primers P3 and P4 also were used to amplify the1665-bp internal genomic region that contains the alternatively splicedtype 1 and 2 cDNA sequences. To confirm the candidate mutation bygenomic sequencing and dot-blot analysis (see below) a 27-mer senseprimer, P7 (5′-AGTAGTCGACATGGGCAGGATGTGTGG-3′) (SEQ ID NO: 17), was usedwith antisense primer P6 to amplify a 567-bp genomic fragment containingthe G-T transversion.

After PCR amplification, the PCR products were isolated from agarosegels and subcloned into either Bluescript KS (+) (Stratagene) or pGEM Zf(−) (Promega) vectors. For each amplified product, from four to sixindependent subclones were sequenced in both orientations by thedideoxynucleotide chain-termination method (Sanger et al., 1977, Proc.Natl. Acad. Sci. U.S.A. 74:5463-5467).

7.1.4. Dot-Blot Analysis

Any nucleotide change that occurred in more than two subcloned PCRproducts was considered a candidate mutation and analyzed by dot-blothybridization with ASOs. In addition, ASOs were constructed and used asprobes to analyze amplified genomic DNA from normal individuals and NPDpatients and family members. From these studies genomic DNA was rapidlyisolated from either whole blood or cultured cells by the followingprocedure. About 0.5 ml of whole blood and 0.5 ml of lysis buffer (10 mMTris/HCl buffer, pH 7.5/5 mM MgCl₂/0.32 M sucrose/1% Triton X-100) weremixed at room temperature. After centrifugation at 13,000×g, thesupernatant was removed, and 0.5 ml of PCR buffer (10 mM Tris HCl,buffer pH 8.3/50 mM KC1/2.5 mM MgCl₂/gelatin at 0.1 mg /ml/0.45%NonidetP40/45% Tween 20/proteinase K at 0.1 mg/ml) was added. Forcultured cells, the lysis step was omitted, and the washed cell pelletswere resuspended directly in PCR buffer (approximately 5×10⁶ cells perml). The samples were then incubated at 60° C. for 1 hour and boiled for10 minutes to inactivate the protease; then 25 μl was removed for PCRamplification. After agarose gel electrophoresis of the PCR products,the concentration of each product was estimated by ethidium bromidestaining. For each sample, approximately 5 μg of DNA was used for thedot-blot analysis.

For the analysis of the R496L mutation, the 567-bp PCR product amplifiedfrom genomic DNA was analyzed by dot-blot hybridization (Sambrook et al,supra) by using Zetabind nylon membrane (AMF Cuno) and a Bio-Raddot-blot apparatus. Hybridizations were performed for at least 3 hoursat 39° C. After hybridization, the blots were washed at room temperaturefor 15 minutes in 6×SSC (1×SSC is 0.15M sodium chloride/0.015 M sodiumcitrate, pH 7.0/0.1% SDS) and then for 2 hr in the same solution ateither 53° C. for the normal (5′-CTATTTGGTACACACGG-3′) (SEQ ID NO: 18)or 48° C. for the mutation-specific (5′-CTATTTGGTACACAAGG-3′) (SEQ IDNO: 19) oligonucleotides.

7.2. Results 7.2.1. Identification of the R496L Mutation in an AshkenaziJewish Type A NPD Patient

To determine the molecular lesions in an Ashkenazi Jewish Type A NPDpatient (proband 1), who had ˜1% of normal ASM activity in culturedfibroblasts, total RNA was isolated from cultured lymphoblasts andreversed-transcribed into cDNA; then the entire coding region was PCRamplified. Nucleotide sequencing of the subcloned PCR products revealeda single point mutation in a CpG dinucleotide, a G-T transversion of nt1487 of the full-length cDNA (FIG. 1). This transversion predicted R496L(SEQ ID NO: 34) in the ASM polypeptide. All other base substitutionsoccurred in only one or two of the subcloned PCR products analyzed.

To confirm the authenticity of this candidate mutation, a 567-bp regionof genomic DNA from the proband, various family members, and 90 normalAshkenazi Jewish individuals was PCR amplified and then hybridized withnormal and R496L-specific radiolabeled oligonucleotides. As shown inFIG. 2C, the PCR-amplified genomic DNA from proband 1 hybridized to themutation-specific, but not to the normal ASO, confirming theauthenticity of the transversibn and indicating that proband 1 washomoallelic for the R496L mutation. Of the nine other family membersstudied, both parents, the paternal grandfather, and a paternal auntwere heterozygous for R496L. The maternal grandmother did not have themutation, suggesting that the maternal grandfather was heterozygous forthis mutation. There was no known consanguinity between the maternal orpaternal grandfathers whose ancestors were from different Europeancountries. Moreover, R496L was not found in any of the 180 ASM allelesstudied from a group of normal Ashkenazi Jewish individuals, indicatingthat the G-T transversion was not a common polymorphism.

7.2.2. Occurrence of R496L in Ashkenazi Jewish and Non-Jewish NPD Type AFamilies

The occurrence of R496L in other NPD families was determined by dot-blotanalysis of genomic DNA from 17 unrelated Ashkenazi Jewish and 18unrelated non-Jewish type A NPD families. As indicated in Table III, ofthe 31 Ashkenazi Jewish Type A NPD alleles studied (i.e., from 6unrelated patients and 19 unrelated obligate heterozygotes), 32% had theR496L mutation. Only proband 1 was homoallelic for the mutation, whereasthree obligate heterozygotes from unrelated families in which materialfrom patients was unavailable were heterozygotes for the R496L mutation.In contrast, only 2 of 36 (5.6%) alleles had the R496L mutation innon-Jewish NPD type A patients, i.e., an American of German ancestry whowas homoallelic.

TABLE III FREQUENCY OF R496L IN ASHKENAZI JEWISH AND NON-JEWISH NPDFAMILIES WITH TYPES A AND B NPD Unrelated Mutant families alleles Sourcestudied, no. studied. no. R496L, % Type A disease Ashkenazi Jewish  17*31 32 Non-Jewish 18 36 5.6 Type B disease Ashkenazi Jewish  2 4 25Non-Jewish 15 30 0.0 *In three of these families only one obligateheterozygous parent was available for analysis.

7.2.3. Occurrence of the R496L Mutation in Ashkenazi Jewish andNon-Jewish NPD Type B Families

Analysis of genomic DNA from two unrelated Ashkenazi Jewish NPD type Bpatients revealed the presence of one R496L allele in one patient(designated proband 2). In contrast, the R496L allele was not found ingenomic DNAs from 15 non-Jewish NPD type B patients (Table III).

7.2.4. Occurrence of the L302P Mutation in Jewish NPD Type A Families

The full-length ASM cDNA was PCR-amplified from a severely affectedAshkenazi Jewish Type A patient, proband 4. The methods for mRNAisolation, reverse transcription and PCR amplification were the same asthose described above for the identification of the R496L mutation. DNAsequencing of the subcloned PCR products from proband 4 revealed asingle T to C transition of nucleotide 905 which predicted thesubstitution of a proline for a leucine at amino acid residue 302 of theASM polypeptide (L302P) (SEQ ID NO: 36). Dot-blot hybridization analysiswith ASOs demonstrated that proband 4 was homoallelic for the L302Pmutation. For the dot-blot analysis, a 606 bp region of the ASM genomicregion is PCR amplified using sense and antisense PCR primers A(5′-TCATCCTCGAGCACTGACCTGCACTGGG-3′) (SEQ ID NO: 31) and B(5′-AGTAGTCGACTGCTAGAGCAATCAGAG-3′) (SEQ ID NO: 25), respectively. Thesequence of the ASOs was L302 (5′-GTCACAGCACTTGTGAG-3′) (SEQ ID NO: 32)and P302 (5′-GTCACAGCACCTGTGAG-3′) (SEQ ID NO: 33). The ASOs werehybridized for at least three hours at 37° C. and then washed for 2hours at 50° C.(L302) and 48° C.(P302). To date, the L302P mutation hasbeen found in about 25% of the ASM alleles studied from Ashkenazi JewishType A patients (8 of 32). In contrast, this lesion has not beenidentified in any non-Jewish type A NPD patients, nor has it been foundin type B NPD patients or normal individuals. Thus, this mutation isspecific to Ashkenazi Jewish individuals with type A NPD and, togetherwith the R496L mutation, over 60% of the mutant ASM alleles in thispopulation can be detected.

7.3. Discussion

Insights into the molecular nature of the remarkably distinct type A andB NPD phenotypes have been gained by the identification of a mutation inthe ASM gene causing this lysosomal storage disease. The G-Ttransversion of coding nt 1487 occurred at a CpG dinucleotide, a knownhotspot for point mutations (Coulondre et al., 1978, Nature 274:775-780)and predicted R496L in the ASM polypeptide. Homoallelism for R496Lresulted in the severe neuronbpathic type A phenotype, as evidenced byproband 1, who had )1% of normal ASM activity. It is not known whetherthe substitution of the basic arginine for the more hydrophobic andneutral leucine residue altered the enzyme polypeptide catalyticactivity, stability, or both, because monospecific anti-human ASMantibodies useful for immunoblotting are not currently available.

Of the 17 unrelated Ashkenazi Jewish type A families studies, 9 wereeither homoallelic or heteroallelic for this lesion. In this sample, thefrequency of the R496L allele was 32%, indicating that this lesion is animportant mutation in type A NPD among Ashkenazi Jewish patients. It islikely that there is another more frequent mutation or, perhaps multiplemutations, causing type A NPD in Ashkenazi Jewish patients. In contrast,analysis of 18 unrelated non-Jewish type A patients revealed thepresence of the R496L allele in only 1 (a frequency of 5.6%). Theoccurrence of the R496L allele in these individuals may have resultedfrom an independent mutational event or the presence of Jewish ancestorsin the non-Jewish families.

One of the two Ashkenazi Jewish type B NPD patients was heteroallelicfor R496L. The other allele in this Jewish type B patient had adifferent ASM mutation, which presumably resulted in the synthesis of apartially functional ASM polypeptide, as this patient had )5% residualASM activity in cultured fibroblasts. The fact that none of the 15non-Jewish type B patients had the R496L allele suggests that thisallele is extremely rare in type B disease outside of the Ashkenazipopulation. That R496L was not a common polymorphism in the AshkenaziJewish population was supported by the fact that it was not found in anyof the 180 ASM alleles analyzed from normal Ashkenazi Jewishindividuals; presumably, this individual was the first NPD heterozygotedetected by molecular screening.

For the past three decades, the genetic mechanisms responsible for thehigh frequency of the mutations that cause Tay-Sachs disease, Gaucherdisease, and NPD in the Ashkenazi Jewish population (gene frequencies ofapproximately 0.02, 0.02, and 0.005, respectively) have been the subjectof interest and debate (Knudson and Kaplan, 1962, CerebralSphingolipidoses, eds. Aronson and Volk, Academic, New York, pp.395-411; Chase and McKusick, 1972, Am. J. Hum. Genet. 24:339-340;Myrianthopoulous et al., 1972, Am. J. Hum. Genet. 24:341-342; Fraikor,1977, Soc. Biol. 24:117-134; Myrianthopoulous and Melnick, 1977, Prog.Clin. Biol. Res. 18:95-196). Intrigued by the fact that all three ofthese disorders are lysosomal diseases resulting from enzymatic defectsin the sphingolipid degradative pathway, investigators suggested thatthere may have been a common selective pressure for their high genefrequencies in the Ashkenazi Jewish population (Myrianthopoulous andMelnick, supra). Others argued that the. higher gene frequencies inAshkenazi Jewish individuals could be due to higher mutation rates forthese genes (Knudson and Kaplan, supra) or founder effect and geneticdrift (Fraikor, supra). The recent identification of the mutationscausing these three diseases in the Ashkenazi Jewish population hasprovided insight into this controversy. To date, three mutations in theβ-hexosaminidase chain (localized to chromosomal region 15q23-24) havebeen identified as the cause of Tay-Sachs disease in almost allAshkenazi Jewish patients. Two of these mutations result in theinfantile form, a 4-bp insertion (Myerowitz and Costigan, 1988, J. Biol.Chem. 263:18587-18589) or a splice-site mutation (Myerowitz, 1988, Proc.Natl. Acad. Sci. U.S.A. 85:3955-3959), which account for approximately80 and 20% of the mutant alleles, respectively. Affected AshkenaziJewish patients with the less frequent and milder chronic or adult-onsetform all have been heteroallelic for a point mutation Gly-Ser atposition 269;) and one of the two infantile-onset alleles. Type 1Gaucher disease among Ashkenazi patients results from multiple mutationsin the β-glucosidase gene (localized to chromosomal region 1q21-q31),the Asn-Ser (at position 370) allele occurring in )75% of the mutantalleles, whereas the other 25% include several other lesions (Tsuji etal., 1987, N. Engl. J.Med. 316:570-757). With the identification of thefirst mutation causing NPD, R496L, it appears that in the AshkenaziJewish population each of those sphingolipidoses results from a commonmutation (i.e., approximately 70% or more of the mutant alleles) and atleast one or more less frequent mutations in their respective genes. Thefact that two or more mutant alleles in each gene occur frequently inthis population argues for selection, rather than for a higher mutationrate or founder effect and genetic drift as the major mechanismresponsible for their increased frequency. Although it is likely thatthe major mutation for each disease first became established in theAshkenazi Jewish population by founder effect and genetic drift, thefinding of two or more mutations in each of these genes supports aselective advantage. Because all three disorders involve defects inlysosomal enzymes that degrade sphingolipids, it is tempting to-suggestthat a common selective agent, such as resistance to an adversesituation (e.g., an infectious disease), could have increased theheterozygote frequency by differential survival (thus, increasedfitness) for individuals heterozygous for each of these disorders.Alternatively, heterozygosity for these mutations may have been selectedfor by unrelated pressures in the past. Although several hypotheses havebeen advanced (Myrianthopoulous and Melnick, supra Myrianthopoulous andAronson, 1972, Advances in Experimental Medicine and Biology, eds., Volkand Aronson, Plenum, New York pp. 561-570), the nature of the selectiveadvantages for these mutations remains unknown.

The identification of the R496L allele and other mutations in the ASMgene in types A and B NPD may provide information for genotype-phenotypecorrelations and permit more accurate genetic counseling for newlydiagnosed cases in families without a previously affected individual.Identification of other mutations, particularly those with residualactivity that cause type B disease, also may provide structure-functioninformation and may facilitate delineation of the active-site region.Previously, the enzymatic detection of heterozygotes for NPD types A andB was not sufficiently reliable to permit mass voluntary screening inthe Ashkenazi Jewish community. Thus, the identification of the R496Land other mutations in types A and B NPD will permit accurateheterozygote identification in families with these lesions as well asheterozygote screening and prevention of NPD in the general Ashkenazi ofJewish population, as has been the prototypic experience with Tay-Sachsdisease (Kaback, 1977, Prog. Clin. Biol. Res. 18:1-7). Using moleculartechniques, we and others (Riggs-Raine, et al., 1990, Engl. J. Med.323:6-12) have already demonstrated the feasibility of molecularheterozygote screening for Tay-Sachs disease in the Ashkenazi Jewishpopulation. The extension of such molecular screening to include themore common mutations causing Gaucher disease and NPD by the use ofmultiplex PCR should permit the simultaneous screening and prevention ofall three sphingolipidoses in the Ashkenazi Jewish population.

8. Example: Niemann-Pick Type B Disease: Identification of a SingleCodon Deletion in the Acid Sphingomyelinase Gene and Genotype/PhenotypeCorrelations in Type A and B Patients 8.1. Materials and Methods 8.1.1.Cell Lines

Primary cultures of fibroblasts and lymphoblasts were established. fromskin biopsies and peripheral blood samples obtained with informedconsent from NPD patients and family members, and from normalindividuals. NPD lines GM00112A, GM00165, GM00370, GM00406, GM00559,GM02895, and GM03252 were obtained from the Human Genetic Mutant CellRepository (Camden, N.J.). Cell lines 444X.F01, 534R.F03, 556X.F01,888V.F01, 2789X.F01, 42.93Q.E02, 4774Z.F01, 5113C.L01, 5115E.F01, and6791M.F01 were obtained from the Service de Biochimie, Hospice de Lyon(Lyon, France). Cell lines DMN 83.126, DMN84.135, DMN 84.87, DMN 86.49,DMN 87.71, DMN 87.99, DMN 88.12, DMN 88.9 and RNS were provided by Dr.Peter Penchev, Developmental and Metabolic Neurology Branch, NationalInstitute of Neurological and Communicative Disorders and Stroke. Thecells were grown in RPMI 1640 media supplemented with 10% fetal bovineserum, 1% penicillin and 1 mg/ml streptomycin by standard procedures(Bernstein et al., 1989 Journal of Clin. Invest. 83: 1390-1399). Thediagnosis of Types A or B NPD was based on clinical criteria (e.g., ageat onset, presence of neurologic involvement, etc.) and by demonstrationof markedly deficient ASM activity in cultured cells (Klar et al., 1988,Clin Chimica Acta 176:259-268). Clinical data on probands 1 and 2 havebeen published (Crocker, 1961, J. Neurochem. 7:69-78; Levran et al., inpress, Proc. Natl. Acad. Sci. U.S.A.), and information on proband 3 wasprovided by Dr. M. Vanier, Department of Biochemistry, Faculte deMedecine, Lyon, France.

8.1.2. Enzyme and Protein Assays

ASM activity was determined in cultured fibroblasts obtained from NPDpatients and normal individuals using the fluorescent natural substrate,[N-12(1-pyrenesulfonyl)amido-dodecanoyl]sphingomyelin(PSA₁₂-sphingomyelin) as previously described (Klar et al., supra). Oneunit (U) of activity equals that amount of enzyme that hydrolyzes onenanomole of substrate per hour. Protein determinations were performed bya modified fluorescamine assay (Bishop and Desnick, 1981, J. Biol. Chem.256:1307-1316).

8.1.3. cDNA and Genomic Amplification and Sequencing

For ASM cDNA amplification and sequencing, total RNA was isolated fromcultured cells by standard procedures (Sambrook et al., 1989, MolecularCloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). First-strand cDNA was synthesized with reversetranscriptase from approximately 5 μg of total RNA using a cDNAsynthesis kit according to the manufacturer's instructions(Boehringer-Mannheim, Indianapolis, Ind.). The ASM cDNA (approximately10% of the total reaction) was PCR-amplified (Saiki et al., 1988,Science 239:487-491) with Taq polymerase (Perkin-Elmer Cetus, Norwalk,Conn.) using the previously described oligonucleotide primers (Levran etal., in press, Proc. Natl. Acad. Sci. U.S.A.). PCR was performed for 30cycles, each consisting of denaturation for 1 minute at 94° C. andannealing and extension for 4 minutes at 72° C. Following amplification,the PCR products were isolated from agarose gels and subcloned intoeither Bluescript KS (+) (Stratagene, La Jolla, Calif.) or pGEM 9Zf (−)(Promega, Madison, Wis.) vectors. For each amplified product, from fourto ten independent subclones were sequenced in both orientations by thedideoxy method (Sanger et al., 1977, Proc. Natl. Acad. Sci. U.S.A.74:5463-5467).

To confirm the candidate mutation, genomic DNA was isolated from theproband (Bernstein et al., 1989, J. Clin. Invest. 83:1390-1399) and a567 bp genomic fragment containing the mutation was PCR-amplified usingsense (5′-AGTAGTCGACATGGGCAGGATGTGTGG-3′) (SEQ ID NO: 17) and antisense(5′-AGTAGTGTCGACTTGCCTGGTTGAACCACAGC-3′) (SEQ ID NO: 16) primerssynthesized on an Applied Biosystems (Foster City, Calif.) Model 380BDNA Synthesizer (Itakura et al., 1984, Ann. Rev. Biochem. 53:323-356).The amplified genomic fragment was isolated, subcloned and sequenced asdescribed above for the PCR-amplified ASM cDNAs.

8.1.4. Dot-Blot Analysis of the Mutant Alleles

For detection of the ΔR608 mutation in other NPD patients, their parentsand relatives, as well as normal individuals, total genomic DNA wasisolated, PCR-amplified as described above, and the 567-bp ASM productwas analyzed by dot-blot hybridization using Zetabind nylon membranes(AMF-Cuno, Meriden, Conn.) and a Bio-Rad (Richmond, Calif.) dot-blotapparatus. Hybridization of the PCR product with the normal ASOs(5′-CTCTGTGCCGCCACCTG-3′) (SEQ ID NO: 20) or with the ΔR608 ASO(5′-GCTCTGTGCCACCTGAT-3′) (SEQ ID NO: 21) were performed for at least 3hours at 39° C. 5′ end labeling of the ASOs with T4 polynucleotidekinase and [γ-32P]ATP (“5000 Ci/mmole) was performed by standardprocedures (Sambrook et al., supra). Following hybridization, the blotswere washed at room temperature for 15 minutes in 6×SSC containing 0.1%SDS, and then 2 h in the same solution at either 54° C. for the normalASO or 50° C. for the ΔR608 ASO. Dot-blot analysis of the R496L mutationwas performed as previously described (Levran et al., supra).

8.2. Results 8.2.1. Identification of the ΔR608 Mutation in an AshkenaziJewish Patient with Type B NPD

Previous studies of an Ashkenazi Jewish Type B NPD patient (proband 2;cell line MS 1271) indicated that he had about 5-8% residual ASMactivity and that he was heteroallelic for the R496L mutation (Levran etal., supra). To identify the molecular lesion in his other ASM allele,total RNA from the proband was reverse-transcribed, the ASM codingregion was PCR-amplified and the PCR products were subcloned intoplasmid vectors for DNA sequencing. A three base deletion (CCG) ofnucleotides 1821 -1823 in the full-length ASM cDNA (Schuchman et al.JBC, in press) was identified which predicted the removal of a singlearginine residue in position 608 of the ASM polypeptide (designatedΔR608). The authenticity of this deletion was determined by genomicsequencing (FIG. 8) and by dot-blot hybridization of PCR-amplifiedgenomic DNA from proband 2 and other family members using an ASOspecific for the ΔR608 (SEQ ID NO: 35) mutation (FIG. 9). In addition,dot-blot hybridizations confirmed the ΔR608/R496L genotype of proband 2,and demonstrated that the ΔR608 and R496L mutations were transmittedfrom his father and mother, respectively. Proband 2's brother did notreceive either mutant ASM allele. The ΔR608 mutation was not identifiedin over 100 ASM alleles from normal individuals, indicated that thedeletion was not a polymorphism.

8.2.2. Occurrence of the ΔR608 Mutation in Types A and B NPD

Table IV shows the occurrence of the ΔR608 mutation in the ASM allelesof other patients and obligate heterozygotes with Types A and B NPD, asdetermined by dot-blot analysis of PCR-amplified genomic DNA.Interestingly, a second, unrelated Ashkenazi Jewish Type B NPD patientwas heteroallelic for the ΔR608 mutation and another, unknown mutant ASMallele. Of the 15 non-Jewish Type B patients studied, only one, an Arabfrom Algeria (proband 3; cell line 534R.F03) was homoallelic for thismutation. This 21 year old male had a mild Type B phenotype (M. T.Vanier, personal communication). Of the 67 ASM alleles from Type A NPDpatients or obligate heterozygotes (26 and 19 unrelated Ashkenazi Jewishand non-Jewish individuals, respectively), none had the ΔR608 mutation.

TABLE IV FREQUENCY OF ΔR608 MUTATION IN ASHKENAZI JEWISH AND NON-JEWISHFAMILIES WITH TYPES A AND B NPD Unrelated Mutant families alleles Sourcestudied studied ΔR608 NPD Type B Ashkenazi Jewish 2 4 50.0 Non-Jewish 1530 6.7 NPD Type A Ashkenazi Jewish 17 31 0.0 Non-Jewish 18 36 0.0

8.2.3. Comparison of the Residual ASM Activities in Type A and B NPDPatients

Table V shows the ASM activities in cultured fibroblasts from probands1, 2 and 3, which were determined using the fluorgenic naturalsubstrate, PSA₁₂-sphingomyelin. Normal individuals had a mean activityof 46.3 nmol cleaved/h/mg. In contrast, Type A proband 1, who washomoallelic for R496L, had less than 1% of normal activity. Type Bproband 2, whose genotype was R496L-ΔR608 had a residual activity ofabout 5% of normal, whereas proband 3 who was homoallelic for ΔR608 hadabout 13% of normal mean ASM activity, indicating that the ΔR608 alleleexpressed functional ASM activity in a dosage dependent manner.

TABLE V GENOTYPE/PHENOTYPE CORRELATIONS IN NIEMANN-PICK TYPES A AND BDISEASE ASM Activity mean Genotype Phenotype (range) % of normal mean(nmol/h/mg) R496L/R496L Type A 0.33 0.7 (proband 1) (0.21-0.47)R496L/ΔR608 Type B 2.23 4.8 (proband 2) (1.8-2.3) ΔR608/ΔR608 Type B5.95 12.8 (proband 3) (5.1-6.9)

The mean ASM activity in three normal individuals was 46.3 nmol/h/mg(range 37.5-61.0 nmol/h/mg).

8.3. Discussion

In 1966, Brady et al. reported that the primary enzymatic defect in TypeA NPD was the deficient activity of ASM (Brady et al., 1966, Proc. Natl.Acad. Sci. USA 55:366-369). In the following year, Schneider and Kennedydemonstrated that ASM activity also was markedly decreased in patientswith the milder, visceral form of NPD now known as Type B disease(Schneider and Kennedy, 1967, J. Lipid. Res. 8:202-206). Subsequentbiochemical analyses of additional patients confirmed these findings(Levade et al., 1986, J. Clin. Chem. Clin. Biochem. 24:205-220; Pouloset al., 1984, Pediat. Res. 18:1088-1092; Besley and Elleder, 1986, J.Inher. Metab. Dis. 9:59-71) and somatic cell genetic studiesdemonstrated that the mutations causing Types A and B disease wereallelic (Besley et al., 1980, Hum. Genet. 54:409-412). These findingsstimulated investigators to speculate that the remarkable clinicalheterogeneity observed among Type A and B NPD patients was due todifferent mutations in the ASM gene which resulted in altered enzymepolypeptides that expressed varying amounts of residual activity (e.g.,Rousson et al., 1986, Immunologic studies on acidic sphingomyelinases.Enzymes of Lipid Metabolism II, New York, Plenum Publishing Corp., NY273-283; Jobb, supra). However, efforts to reliably predict either thedisease subtype or the severity of Type B patients by the amount ofresidual ASM activity have not been possible, in part due to theinability of assay procedures to accurately distinguish between patientswith low levels of residual ASM activity and/or the presence of theneutral sphingomyelinase activity in cell homogenates (Chatterjee andGosh, 1989, J. Biol. Chem. 264:12,554-12,561). In addition, theinability to reliably discriminate obligate heterozygotes for Types Aand B NPD from non-carriers by the determination of ASM activity inisolated leukocytes has precluded carrier screening for NPD.

The recent cloning and sequencing of the ASM cDNA (Quintern et al.,supra; Schuchman et al., in press, J. Biol. Chem.) has permittedidentification of the first mutations which result in Types A and B NPD.Previously, the R496L mutation, due to a single G to T transversion, wasfound in 32% of the Ashkenazi Jewish Type A alleles studied. Incontrast, it was present in only 5.6% of the non-Jewish Type A allelesanalyzed (Levran et al., supra). In addition, proband 2, an AshkenaziJewish Type B NPD patient was found to be heteroallelic for the R496Lmutation. In the present study, a three base deletion (ΔR608) in the ASMgene was identified as the other mutation in proband 2. As shown in FIG.10B, the three base deletion, CCG, removed the last nucleotide of codonCys-607 (TGC) and the first two nucleotides of codon Arg-608 (GCC). Thenew codon 607 formed by this deletion, TGC, also encoded a cysteineresidue. Thus, the deletion resulted in the elimination of a singlecodon, 608, which encoded an arginine residue.

Detection of the R496L and ΔR608 mutations in patients with Types A andB NPD has permitted genotype/phenotype correlations and providedinsights into the function of the altered enzymes encoded by thesemutant alleles (Table II). The homoallelic (R496L/ΔR608) Type A patientwho expresses less than 1% of normal ASM activity in culturedfibroblasts indicates that the R496L mutation encodes an ASM polypeptidewith little, if any, catalytic activity and/or markedly decreasedstability, thereby resulting the neuronopathic phenotype. In contrast,both heteroallelic (R496L/ΔR608) and homoallelic (ΔR608/ΔR608) Type Bpatients express an enzyme with sufficient residual activity to preventneurologic manifestations. These findings suggest that the substitutionof an arginine for a leucine at position 496 was more damaging to theenzyme's activity and/or stability than the deletion of an arginineresidue in position 608. It follows that Type B patients who arehomoallelic for ΔR608 would have a milder disease course than Type Bpatients who are heteroallelic for ΔR608 and a Type A mutation. Notably,proband 3 had comparatively milder disease manifestations than proband 2at the same age. Thus, these genotype/phenotype correlations indicatethat the more residual ASM activity expressed by the mutant alleles, themilder the disease manifestations. Ideally, in vitro expression wouldpermit comparisons of the activity and stability of the residual enzymesexpressed by each mutant ASM allele. However, it is likely that theresidual activities expressed by the mutant alleles would be too low topermit biochemical characterization, particularly since eukaryoticexpression systems (e.g., COS-1 cells, CHO cells) have endogenous ASMactivity and prokaryotic systems do not perform the post-translationalmodifications (e.g., N-glycosylation) required for ASM activity.

The occurrence of genetic heterogeneity resulting in dramaticallydifferent phenotypes is a hallmark of the lysosomal storage diseases. Anotable example in which the molecular lesions have been correlated withdistinct phenotypes is Tay-Sachs disease (Neufeld, 1989, J. Biol. Chem.264:10,927-10,930; Navon and Proia, 1989, Science 243:1471-1474).Mutations causing the classic, infantile form of Tay-Sachs disease haveincluded deletions and splice site mutations in the β-hexosaminidase Aα-chain gene that resulted in no detectable transcripts, whereas anexonic point mutation expresses sufficient enzymatic activity to resultin the adult or chronic type of GM₂ gangliosidosis. Analogously,identification of the R496L and ΔR608 mutations has provided the firstinsights into the molecular lesions in the ASM gene underlying theremarkable phenotypic heterogeneity in NPD. Clearly, individuals who arehomoallelic for the R496L mutation will have a severe neuronopathicphenotype and Type A NPD. The phenotype of patients heteroallelic forR496L will depend on the genetic lesion present on their other ASMallele. In contrast, patients who are either homo- or heteroallelic forthe ΔR608 (e.g., probands 2 and 3) will most likely have Type B NPD. Theidentification of additional mutations causing Types A and B NPD shouldpermit reliable genotype/phenotype correlations and provide furtherinsights into the functional organization of the ASM polypeptide.

Various references are cited herein that are hereby incorporated byreferences in their entirety.

36 2347 base pairs nucleic acid single unknown cDNA CDS 88..1977 1GGCGCCGCCC GGGGCCCTGA GGGCTGGCTA GGGTCCAGGC CGGGGGGGAC GGGACAGACG 60AACCAGCCCC GTGTAGGAAG CGCGACA ATG CCC CGC TAC GGA GCG TCA CTC 111 MetPro Arg Tyr Gly Ala Ser Leu 1 5 CGC CAG AGC TGC CCC AGG TCC GGC CGG GAGCAG GGA CAA GAC GGG ACC 159 Arg Gln Ser Cys Pro Arg Ser Gly Arg Glu GlnGly Gln Asp Gly Thr 10 15 20 GCC GGA GCC CCC GGA CTC CTT TGG ATG GGC CTGGTG CTG GCG CTG GCG 207 Ala Gly Ala Pro Gly Leu Leu Trp Met Gly Leu ValLeu Ala Leu Ala 25 30 35 40 CTG GCG CTG GCG CTG GCT CTG TCT GAC TCT CGGGTT CTC TGG GCT CCG 255 Leu Ala Leu Ala Leu Ala Leu Ser Asp Ser Arg ValLeu Trp Ala Pro 45 50 55 GCA GAG GCT CAC CCT CTT TCT CCC CAA GGC CAT CCTGCC AGG TTA CAT 303 Ala Glu Ala His Pro Leu Ser Pro Gln Gly His Pro AlaArg Leu His 60 65 70 CGC ATA GTG CCC CGG CTC CGA GAT GTC TTT GGG TGG GGGAAC CTC ACC 351 Arg Ile Val Pro Arg Leu Arg Asp Val Phe Gly Trp Gly AsnLeu Thr 75 80 85 TGC CCA ATC TGC AAA GGT CTA TTC ACC GCC ATC AAC CTC GGGCTG AAG 399 Cys Pro Ile Cys Lys Gly Leu Phe Thr Ala Ile Asn Leu Gly LeuLys 90 95 100 AAG GAA CCC AAT GTG GCT CGC GTG GGC TCC GTG GCC ATC AAGCTG TGC 447 Lys Glu Pro Asn Val Ala Arg Val Gly Ser Val Ala Ile Lys LeuCys 105 110 115 120 AAT CTG CTG AAG ATA GCA CCA CCT GCC GTG TGC CAA TCCATT GTC CAC 495 Asn Leu Leu Lys Ile Ala Pro Pro Ala Val Cys Gln Ser IleVal His 125 130 135 CTC TTT GAG GAT GAC ATG GTG GAG GTG TGG AGA CGC TCAGTG CTG AGC 543 Leu Phe Glu Asp Asp Met Val Glu Val Trp Arg Arg Ser ValLeu Ser 140 145 150 CCA TCT GAG GCC TGT GGC CTG CTC CTG GGC TCC ACC TGTGGG CAC TGG 591 Pro Ser Glu Ala Cys Gly Leu Leu Leu Gly Ser Thr Cys GlyHis Trp 155 160 165 GAC ATT TTC TCA TCT TGG AAC ATC TCT TTG CCT ACT GTGCCG AAG CCG 639 Asp Ile Phe Ser Ser Trp Asn Ile Ser Leu Pro Thr Val ProLys Pro 170 175 180 CCC CCC AAA CCC CCT AGC CCC CCA GCC CCA GGT GCC CCTGTC AGC CGC 687 Pro Pro Lys Pro Pro Ser Pro Pro Ala Pro Gly Ala Pro ValSer Arg 185 190 195 200 ATC CTC TTC CTC ACT GAC CTG CAC TGG GAT CAT GACTAC CTG GAG GGC 735 Ile Leu Phe Leu Thr Asp Leu His Trp Asp His Asp TyrLeu Glu Gly 205 210 215 ACG GAC CCT GAC TGT GCA GAC CCA CTG TGC TGC CGCCGG GGT TCT GGC 783 Thr Asp Pro Asp Cys Ala Asp Pro Leu Cys Cys Arg ArgGly Ser Gly 220 225 230 CTG CCG CCC GCA TCC CGG CCA GGT GCC GGA TAC TGGGGC GAA TAC AGC 831 Leu Pro Pro Ala Ser Arg Pro Gly Ala Gly Tyr Trp GlyGlu Tyr Ser 235 240 245 AAG TGT GAC CTG CCC CTG AGG ACC CTG GAG AGC CTGTTG AGT GGG CTG 879 Lys Cys Asp Leu Pro Leu Arg Thr Leu Glu Ser Leu LeuSer Gly Leu 250 255 260 GGC CCA GCC GGC CCT TTT GAT ATG GTG TAC TGG ACAGGA GAC ATC CCC 927 Gly Pro Ala Gly Pro Phe Asp Met Val Tyr Trp Thr GlyAsp Ile Pro 265 270 275 280 GCA CAT GAT GTC TGG CAC CAG ACT CGT CAG GACCAA CTG CGG GCC CTG 975 Ala His Asp Val Trp His Gln Thr Arg Gln Asp GlnLeu Arg Ala Leu 285 290 295 ACC ACC GTC ACA GCA CTT GTG AGG AAG TTC CTGGGG CCA GTG CCA GTG 1023 Thr Thr Val Thr Ala Leu Val Arg Lys Phe Leu GlyPro Val Pro Val 300 305 310 TAC CCT GCT GTG GGT AAC CAT GAA AGC ATA CCTGTC AAT AGC TTC CCT 1071 Tyr Pro Ala Val Gly Asn His Glu Ser Ile Pro ValAsn Ser Phe Pro 315 320 325 CCC CCC TTC ATT GAG GGC AAC CAC TCC TCC CGCTGG CTC TAT GAA GCG 1119 Pro Pro Phe Ile Glu Gly Asn His Ser Ser Arg TrpLeu Tyr Glu Ala 330 335 340 ATG GCC AAG GCT TGG GAG CCC TGG CTG CCT GCCGAA GCC CTG CGC ACC 1167 Met Ala Lys Ala Trp Glu Pro Trp Leu Pro Ala GluAla Leu Arg Thr 345 350 355 360 CTC AGA ATT GGG GGG TTC TAT GCT CTT TCCCCA TAC CCC GGT CTC CGC 1215 Leu Arg Ile Gly Gly Phe Tyr Ala Leu Ser ProTyr Pro Gly Leu Arg 365 370 375 CTC ATC TCT CTC AAT ATG AAT TTT TGT TCCCGT GAG AAC TTC TGG CTC 1263 Leu Ile Ser Leu Asn Met Asn Phe Cys Ser ArgGlu Asn Phe Trp Leu 380 385 390 TTG ATC AAC TCC ACG GAT CCC GCA GGA CAGCTC CAG TGG CTG GTG GGG 1311 Leu Ile Asn Ser Thr Asp Pro Ala Gly Gln LeuGln Trp Leu Val Gly 395 400 405 GAG CTT CAG GCT GCT GAG GAT CGA GGA GACAAA GTG CAT ATA ATT GGC 1359 Glu Leu Gln Ala Ala Glu Asp Arg Gly Asp LysVal His Ile Ile Gly 410 415 420 CAC ATT CCC CCA GGG CAC TGT CTG AAG AGCTGG AGC TGG AAT TAT TAC 1407 His Ile Pro Pro Gly His Cys Leu Lys Ser TrpSer Trp Asn Tyr Tyr 425 430 435 440 CGA ATT GTA GCC AGG TAT GAG AAC ACCCTG GCT GCT CAG TTC TTT GGC 1455 Arg Ile Val Ala Arg Tyr Glu Asn Thr LeuAla Ala Gln Phe Phe Gly 445 450 455 CAC ACT CAT GTG GAT GAA TTT GAG GTCTTC TAT GAT GAA GAG ACT CTG 1503 His Thr His Val Asp Glu Phe Glu Val PheTyr Asp Glu Glu Thr Leu 460 465 470 AGC CGG CCG CTG GCT GTA GCC TTC CTGGCA CCC AGT GCA ACT ACC TAC 1551 Ser Arg Pro Leu Ala Val Ala Phe Leu AlaPro Ser Ala Thr Thr Tyr 475 480 485 ATC GGC CTT AAT CCT GGT TAC CGT GTGTAC CAA ATA GAT GGA AAC TAC 1599 Ile Gly Leu Asn Pro Gly Tyr Arg Val TyrGln Ile Asp Gly Asn Tyr 490 495 500 TCC AGG AGC TCT CAC GTG GTC CTG GACCAT GAG ACC TAC ATC CTG AAT 1647 Ser Arg Ser Ser His Val Val Leu Asp HisGlu Thr Tyr Ile Leu Asn 505 510 515 520 CTG ACC CAG GCA AAC ATA CCG GGAGCC ATA CCG CAC TGG CAG CTT CTC 1695 Leu Thr Gln Ala Asn Ile Pro Gly AlaIle Pro His Trp Gln Leu Leu 525 530 535 TAC AGG GCT CGA GAA ACC TAT GGGCTG CCC AAC ACA CTG CCT ACC GCC 1743 Tyr Arg Ala Arg Glu Thr Tyr Gly LeuPro Asn Thr Leu Pro Thr Ala 540 545 550 TGG CAC AAC CTG GTA TAT CGC ATGCGG GGC GAC ATG CAA CTT TTC CAG 1791 Trp His Asn Leu Val Tyr Arg Met ArgGly Asp Met Gln Leu Phe Gln 555 560 565 ACC TTC TGG TTT CTC TAC CAT AAGGGC CAC CCA CCC TCG GAG CCC TGT 1839 Thr Phe Trp Phe Leu Tyr His Lys GlyHis Pro Pro Ser Glu Pro Cys 570 575 580 GGC ACG CCC TGC CGT CTG GCT ACTCTT TGT GCC CAG CTC TCT GCC CGT 1887 Gly Thr Pro Cys Arg Leu Ala Thr LeuCys Ala Gln Leu Ser Ala Arg 585 590 595 600 GCT GAC AGC CCT GCT CTG TGCCGC CAC CTG ATG CCA GAT GGG AGC CTC 1935 Ala Asp Ser Pro Ala Leu Cys ArgHis Leu Met Pro Asp Gly Ser Leu 605 610 615 CCA GAG GCC CAG AGC CTG TGGCCA AGG CCA CTG TTT TGC TAGGGCCCCA 1984 Pro Glu Ala Gln Ser Leu Trp ProArg Pro Leu Phe Cys 620 625 630 GGGCCCACAT TTGGGAAAGT TCTTGATGTAGGAAAGGGTG AAAAAGCCCA AATGCTGCTG 2044 TGGTTCAACC AGGCAAGATC ATCCGGTGAAAGAACCAGTC CCTGGGCCCC AAGGATGCCG 2104 GGGAAACAGG ACCTTCTCCT TTCCTGGAGCTGGTTTAGCT GGATATGGGA GGGGGTTTGG 2164 CTGCCTGTGC CCAGGAGCTA GACTGCCTTGAGGCTGCTGT CCTTTCACAG CCATGGAGTA 2224 GAGGCCTAAG TTGACACTGC CCTGGGCAGACAAGACAGGA GCTGTCGCCC CAGGCCTGTG 2284 CTGCCCAGCC AGGAACCCTG TACTGCTGCTGCGACCTGAT GCTGCCAGTC TGTTAAAATA 2344 AAG 2347 629 amino acids aminoacid unknown protein 2 Met Pro Arg Tyr Gly Ala Ser Leu Arg Gln Ser CysPro Arg Ser Gly 1 5 10 15 Arg Glu Gln Gly Gln Asp Gly Thr Ala Gly AlaPro Gly Leu Leu Trp 20 25 30 Met Gly Leu Val Leu Ala Leu Ala Leu Ala LeuAla Leu Ala Leu Ser 35 40 45 Asp Ser Arg Val Leu Trp Ala Pro Ala Glu AlaHis Pro Leu Ser Pro 50 55 60 Gln Gly His Pro Ala Arg Leu His Arg Ile ValPro Arg Leu Arg Asp 65 70 75 80 Val Phe Gly Trp Gly Asn Leu Thr Cys ProIle Cys Lys Gly Leu Phe 85 90 95 Thr Ala Ile Asn Leu Gly Leu Lys Lys GluPro Asn Val Ala Arg Val 100 105 110 Gly Ser Val Ala Ile Lys Leu Cys AsnLeu Leu Lys Ile Ala Pro Pro 115 120 125 Ala Val Cys Gln Ser Ile Val HisLeu Phe Glu Asp Asp Met Val Glu 130 135 140 Val Trp Arg Arg Ser Val LeuSer Pro Ser Glu Ala Cys Gly Leu Leu 145 150 155 160 Leu Gly Ser Thr CysGly His Trp Asp Ile Phe Ser Ser Trp Asn Ile 165 170 175 Ser Leu Pro ThrVal Pro Lys Pro Pro Pro Lys Pro Pro Ser Pro Pro 180 185 190 Ala Pro GlyAla Pro Val Ser Arg Ile Leu Phe Leu Thr Asp Leu His 195 200 205 Trp AspHis Asp Tyr Leu Glu Gly Thr Asp Pro Asp Cys Ala Asp Pro 210 215 220 LeuCys Cys Arg Arg Gly Ser Gly Leu Pro Pro Ala Ser Arg Pro Gly 225 230 235240 Ala Gly Tyr Trp Gly Glu Tyr Ser Lys Cys Asp Leu Pro Leu Arg Thr 245250 255 Leu Glu Ser Leu Leu Ser Gly Leu Gly Pro Ala Gly Pro Phe Asp Met260 265 270 Val Tyr Trp Thr Gly Asp Ile Pro Ala His Asp Val Trp His GlnThr 275 280 285 Arg Gln Asp Gln Leu Arg Ala Leu Thr Thr Val Thr Ala LeuVal Arg 290 295 300 Lys Phe Leu Gly Pro Val Pro Val Tyr Pro Ala Val GlyAsn His Glu 305 310 315 320 Ser Ile Pro Val Asn Ser Phe Pro Pro Pro PheIle Glu Gly Asn His 325 330 335 Ser Ser Arg Trp Leu Tyr Glu Ala Met AlaLys Ala Trp Glu Pro Trp 340 345 350 Leu Pro Ala Glu Ala Leu Arg Thr LeuArg Ile Gly Gly Phe Tyr Ala 355 360 365 Leu Ser Pro Tyr Pro Gly Leu ArgLeu Ile Ser Leu Asn Met Asn Phe 370 375 380 Cys Ser Arg Glu Asn Phe TrpLeu Leu Ile Asn Ser Thr Asp Pro Ala 385 390 395 400 Gly Gln Leu Gln TrpLeu Val Gly Glu Leu Gln Ala Ala Glu Asp Arg 405 410 415 Gly Asp Lys ValHis Ile Ile Gly His Ile Pro Pro Gly His Cys Leu 420 425 430 Lys Ser TrpSer Trp Asn Tyr Tyr Arg Ile Val Ala Arg Tyr Glu Asn 435 440 445 Thr LeuAla Ala Gln Phe Phe Gly His Thr His Val Asp Glu Phe Glu 450 455 460 ValPhe Tyr Asp Glu Glu Thr Leu Ser Arg Pro Leu Ala Val Ala Phe 465 470 475480 Leu Ala Pro Ser Ala Thr Thr Tyr Ile Gly Leu Asn Pro Gly Tyr Arg 485490 495 Val Tyr Gln Ile Asp Gly Asn Tyr Ser Arg Ser Ser His Val Val Leu500 505 510 Asp His Glu Thr Tyr Ile Leu Asn Leu Thr Gln Ala Asn Ile ProGly 515 520 525 Ala Ile Pro His Trp Gln Leu Leu Tyr Arg Ala Arg Glu ThrTyr Gly 530 535 540 Leu Pro Asn Thr Leu Pro Thr Ala Trp His Asn Leu ValTyr Arg Met 545 550 555 560 Arg Gly Asp Met Gln Leu Phe Gln Thr Phe TrpPhe Leu Tyr His Lys 565 570 575 Gly His Pro Pro Ser Glu Pro Cys Gly ThrPro Cys Arg Leu Ala Thr 580 585 590 Leu Cys Ala Gln Leu Ser Ala Arg AlaAsp Ser Pro Ala Leu Cys Arg 595 600 605 His Leu Met Pro Asp Gly Ser LeuPro Glu Ala Gln Ser Leu Trp Pro 610 615 620 Arg Pro Leu Phe Cys 625 1664base pairs nucleic acid single unknown cDNA 3 TGGGTAACCA TGAAAGCACACCTGTCAATA GCTTCCCTCC CCCCTTCATT GAGGGCAACC 60 ACTCCTCCCG CTGGCTCTATGAAGCGATGG CCAAGGCTTG GGAGCCCTGG CTGCCTGCCG 120 AAGCCCTGCG CACCTCAGGTACTTATCGTC CGTGGAAACC CAGGAAGGGA AAAGAAAGGT 180 GAATGAAAGT GAAGGGAGAAGGGAACCTGG GGCATTGTCT CTGATTGCTC TAGCATGAGT 240 CCTTAGTGCT CTTCATTTGGCTCCCCTAAT CTGACTCCTC CTTCCCTTTC TACTGTTTTG 300 CCGCACCAGG CTTTTTTTTTTTTTTTTTTT TAGCTTTAGT TTTTGTAGAG ACAAGATCTT 360 GCTATGTTGC CCAGGCTGGTCTCAAACACC TAACCTCAAG CAATCCTCCC GCCTCGGCCT 420 CCCAAAATGC TGGGCACAGGCATCAGCTAC TGCTCCTGGC CCTCCCTTTT TTTTTTTTTT 480 TTTTTTTTTG AGATGGAATCTTGCTCTGTT GCCCAGGCTG GAGTGCAGTG GCAACCATCT 540 CAGCTCACTA CAGCCTCCACCTCCTGGGTT CAAGCAATTC TGCCTCAGCC TCCCAAGTAC 600 CTGGGACTAC AGGTGCACGCCACCACACCC AGCTAATTTT TGTATTTTTA GTAGAGATGG 660 GGTTTCACCA TGTTGGCCAAGATGGTCTTG ATCTCCTGAC CTCATGATCT GCCCACCTCG 720 GCCTCCCAAA GTGCTGGGATTACAGGCATG AACCACTGCA CCCAGCTTTC CAGCCCTCCC 780 TTTCTACTCT TATCTCCAGCCACCCTCCTT CAAAGGTCTG GCAGCATAAC CTCTCTATGC 840 CCCAGCTGTG TCTTTGCTCATATTGGCCCT CTGGAAATGA TTTCCCCCTT TTTTTTAAGT 900 GCTCCAGTTT TTCCCACCTTATCCATCCCA TGTCATCTTC CCTCTGTGTG GTCCTTGCTT 960 CCCATTCTAG CTAACTCTTATCCCTCCCCC ATACTCCTGG AGCCCTCTGC CCTCAGAGTC 1020 TTTTGTGTCA CACAGACCCAATAATTAGAA CTGTTTGGTC TCTGGCTAGA CTGTGAGCTC 1080 CTTGCAGGTG GGGAAGATGTCATGTATGCT TTTACCCTCC ACCCAAATGC CCAGCACAGG 1140 AGGACCAGGA TTGGAACAAGTGTTGACCTC TCATGTTTAC TTTGTTTCAG AATTGGGGGG 1200 TTCTATGCTC TTTCCCCATACCCCGGTCTC CGCCTCATCT CTCTCAATAT GAATTTTTGT 1260 TCCCGTGAGA ACTTCTGGCTCTTGATCAAC TCCACGGATC CCGCAGGACA GCTCCAGTGG 1320 CTGGTGGGGG AGCTTCAGGCTGCTGAGGAT CGAGGAGACA AAGTGAGGGC CAGTAGTGGG 1380 AACACGGTGG TGCTGGGGGACAAGCAGGCT CCTGTTGAGC TGGAGCACCT CTGGGCACAG 1440 AAGTTTTATT TTCCTGGCATTCCCAACAAG TGTTCCCTGG GGATTCAGCT CATGGTCACT 1500 GTTGAAAGCC TTCATTCAGTCCCCCTTTCT CTAGCCAGGG CTGCCTGGAC CCCTGGATGC 1560 CCTGATTACC ATCCTTAATTCTCCCTACTA GGTGCATATA ATTGGCCACA TTCCCCCAGG 1620 GCACTGTCTG AAGAGCTGGAGCTGGAATTA TTACCGAATT GTGA 1664 4741 base pairs nucleic acid singleunknown DNA (genomic) 4 TCGACAGCCG CCCGCCACCG AGAGATCAGC TGTCAGAGATCAGAGGAAGA GGAAGGGGCG 60 GAGCTGCTTT GCGGCCGGCC GGAGCAGTCA GCCGACTACAGAGAAGGGTA ATCGGGTGTC 120 CCCGGCGCCG CCCGGGGCCC TGAGGGCTGG CTAGGGTCCAGGCCGGGGGG GACGGGACAG 180 ACGAACCAGC CCCGTGTAGG AAGCGCGACA ATGCCCCGCTACGGAGCGTC ACTCCGCCAG 240 AGCTGCCCCA GGTCCGGCCG GGAGCAGGGA CAAGACGGGACCGCCGGAGC CCCCGGACTC 300 CTTTGGATGG GCCTGGCGCT GGCGCTGGCG CTGGCGCTGGCGCTGGCTCT GTCTGACTCT 360 CGGGTTCTCT GGGCTCCGGC AGAGGCTCAC CCTCTTTCTCCCCAAGGCCA TCCTGCCAGG 420 TTACATCGCA TAGTGCCCCG GCTCCGAGAT GTCTTTGGGTGGGGGAACCT CACCTGCCCA 480 ATCTGCAAAG GTCTATTCAC CGCCATCAAC CTCGGGCTGAAGGTGAGCAC TGAAGGGGCT 540 GCAGTGGAGG AGGCCGAAAG GAGTGCTGGG GCTGGGGGCTGGGGCTGATG CTGGTGCGCT 600 GGGCTCAGAA TGCATCCCTG ATGGAGAGGG TGGCATCTACAATCCATCAC TGAGTTTGCT 660 CCCCTTTGGG GACACCCATG GCTACATGCC ACCATCACCCCATTGTGACC TTTGTGAAGT 720 AAGAAATAAT GCAGACAGTG CCTGAGGAAG TCAGCTTGCCAAGCAAAGGC CTCATGCCAC 780 AGGCCGCTGA GCTAAAGAAG AAGCGATGGC CTGGTGCTGCCTGAGTTACA GGGCAATATC 840 TGGAAGGCAA AGGTGTGCAC TGAGCTTGGT GCACTGAGTCCTGCCCAGCC CCAGTTTGGA 900 AATGGAGGCC AAGGGGTGGT GGCCAGGGGT TGGCCTGGTTCCTCTGCTCT GCCTCTGATT 960 TCTCACCATG CGCTCCTCCC ACTGCAGAAG GAACCCAATGTGGCTCGCGT GGGCTCCGTG 1020 GCCATCAAGC TGTGCAATCT GCTGAAGATA GCACCACCTGCCGTGTGCCA ATCCATTGTC 1080 CACCTCTTTG AGGATGACAT GGTGGAGGTG TGGAGACGCTCAGTGCTGAG CCCATCTGAG 1140 GCCTGTGGCC TGCTCCTGGG CTCCACCTGT GGGCACTGGGACATTTTCTC ATCTTGGAAC 1200 CGGACACCGG ACGAGGACCC GAGGTGGACA CCCGTGACCCTGTAAAAGAG TAGAACCTTG 1260 ATCTCTTTGC CTACTGTGCC GAAGCCGCCC CCCAAACCCCCTAGCCCCCC AGCCCCAGGT 1320 GCCCCTGTCA GCCGCATCCT CTTCCTCACT GACCTGCACTGGGATCATGA CTACCTGGAG 1380 GGCACGGACC CTGACTGTGC AGACCCACTG TGCTGCCGCCGGGGTTCTGG CCTGCCGCCC 1440 GCATCCCGGC CAGGTGCCGG ATACTGGGGC GAATACAGCAAGTGTGACCT GCCCCTGAGG 1500 ACCCTGGAGA GCCTGTTGAG TGGGCTGGGC CCAGCCGGCCCTTTTGATAT GGTGTACTGG 1560 ACAGGAGACA TCCCCGCACA TGATGTCTGG CACCAGACTCGTCAGGACCA ACTGCGGGCC 1620 CTGACCACCG TCACAGCACT TGTGAGGAAG TTCCTGGGGCCAGTGCCAGT GTACCCTGCT 1680 GTGGGTAACC ATGAAAGCAC ACCTGTCAAT AGCTTCCCTCCCCCCTTCAT TGAGGGCAAC 1740 CACTCCTCCC GCTGGCTCTA TGAAGCGATG GCCAAGGCTTGGGAGCCCTG GCTGCCTGCC 1800 GAAGCCCTGC GCACCCTCAG GTACTTATCG TCCGTGGAAACCCAGGAAGG GAAAAGAAAG 1860 GTGAATGAAA GTGAAGGGAG AAGGGAACCT GGGGCATTGTCTCTGATTGC TCTAGCATGA 1920 GTCCTTAGTG CTCTTCATTT GGCTCCCCTA ATCTGACTCCTCCTTCCCTT TCTACTGTTT 1980 TGCCGCACCA GGCTTTTTTT TTTTTTTTTT TTTTAGTTTAGTTTTTGTAG AGACAAGATC 2040 TTGCTATGTT GCCCAGGCTG GTCTCAAACA CCTAACCTCAAGCAATCCTC CCGCCTCGGC 2100 CTCCCAAAAT GCTGGGACCA CAGGCATCAG CTACTGCTCCTGGCCCTCCC TTTTTTTTTT 2160 TTTTTTTTTT TTTTTTTTTT GAGATGGAAT CTTGCTCTGTTGCCCAGGCT GGAGTGCAGT 2220 GGCACCATCT CAGCTCACTA CAGCCTCCAC CTCCTGGGTTCAAGCAATTC TGCCTCAGCC 2280 TCCCAAGTAC CTGGGACTAC AGGTGCACGC CACCACACCCAGCTAATTTT TGTATTTTTA 2340 GTAGAGATGG GGTTTCACCA TGTTGGCCAA GATGGTCTTGATCTCCTGAC CTCATGATCT 2400 GCCCACCTCG GCCTCCCAAA GTGCTGGGAT TACAGGCATGAACCACTGCA CCCAGCTTTC 2460 CAGCCCTCCC TTTCTACTCT TATCTCCAGC CACCCTCCTTCAAAGGTCTG GCAGCATAAC 2520 CTCTCTATGC CCCAGCTGTG TCTTTGCTCA TGTTGGCCCTCTGGAAATGA TTTCCCCCTT 2580 TTTTTTAAGT GCTCCAGTTT TTCCCACCTT ATCCATCCCATGTCATCTTC CCTCTGTGTG 2640 GTCCTTGCTT CCCATTCTAG CTAACTCTTA TCCCTCCCCCATACTCCTGG AGCCCTCTGC 2700 CCTCAGATGC TTTTGTGTCA CACAGACCCA ATAATTAGAACTGTTTGGTC TCTGGCTAGA 2760 CTGTGAGCTC CTTGCAGGTG GGGAAGATGT CATGTATGCTTTTACCCTCC ACCCAAATGC 2820 CCAGCACAGG AGGACCAGGA TTGGAACAAG TGTTGACCTCTCATGTTTAC TTTGTTTCAG 2880 AATTGGGGGG TTCTATGCTC TTTCCCCATA CCCCGGTCTCCGCCTCATCT CTCTCAATAT 2940 GAATTTTTGT TCCCGTGAGA ACTTCTGGCT CTTGATCAACTCCACGGATC CCGCAGGACA 3000 GCTCCAGTGG CTGGTGGGGG AGCTTCAGGC TGCTGAGGATCGAGGAGACA AAGTGAGGGC 3060 CAGTAGTGGG AACACGGTGG TGCTGGGGGA CAAGCAGGCTCCTGTTGAGC TGGAGCACCT 3120 CTGGGCACAG AAGTTTTATT TTCCTGGCAT TCCCAACAAGTGTTCCCTGG GGATTCAGCT 3180 CATGGTCACT GTTGAAAGCC TTCATTCAGT CCCCCTTTCTCTAGCCAGGG CTGCCTGGAC 3240 CCCTGGATGC CCTGATTACC ATCCTTAATT CTCCCTACTAGGTGCATATA ATTGGCCACA 3300 TTCCCCCAGG GCACTGTCTG AAGAGCTGGA GCTGGAATTATTACCGAATT GTAGCCAGGT 3360 AGGACGGAGA TGAGGGTGGG AATAGGGACA GGGTGAGTGTCTGAAGGCTG AAAATTCCCT 3420 TGAGCATCTC ACCATCCCTG TTGTCCCATG GAGTGGGGAGGCTCCTCACT AGAACAGGTT 3480 GGAGAAAGAG GGCATCCTAT CTCCCCAGAT GTCTTCCTACCCCTCCCTAG AATCTTCTGA 3540 ATGTAGTACC TTCTGGCCAG GTATGAGAAC ACCCTGGCTGCTCAGTTCTT TGGCCACACT 3600 CATGTGGATG AATTTGAGGT CTTCTATGAT GAAGAGACTCTGAGCCGGCC GCTGGCTGTA 3660 GCCTTCCTGG CACCCAGTGC AACTACCTAC ATCGGCCTTAATCCTGGTGA GTGAGGCAGA 3720 AGGGAGCCTC CCTTATCCTG GAGTTGGTGG GATAGGGGAAGGAGGTTGGA GCCAGAGCCT 3780 GCAAAGCATG GGCAGGATGT GTGGCCCCTC CCTGGAGTTACCCTTGCTCC TTGCCCCTCC 3840 AGTCAGCCCC ACATCCTTGC AGGTTACCGT GTGTACCAAATAGATGGAAA CTACTCCGGG 3900 AGCTCTCACG TGGTCCTGGA CCATGAGACC TACATCCTGAATCTGACCCA GGCAAACATA 3960 CCGGGAGCCA TACCGCACTG GCAGCTTCTC TACAGGGCTCGAGAAACCTA TGGGCTGCCC 4020 AACACACTGC CTACCGCCTG GCACAACCTG GTATATCGCATGCGGGGCGA CATGCAACTT 4080 TTCCAGACCT TCTGGTTTCT CTACCATAAG GGCCACCCACCCTCGGAGCC CTGTGGCACG 4140 CCCTGCCGTC TGGCTACTCT TTGTGCCCAG CTCTCTGCCCGTGCTGACAG CCCTGCTCTG 4200 TGCCGCCACC TGATGCCAGA TGGGAGCCTC CCAGAGGCCCAGAGCCTGTG GCCAAGGCCA 4260 CTGTTTTGCT AGGGCCCCAG GGCCCACATT TGGGAAAGTTCTTGATGTAG GAAAGGGTGA 4320 AAAAGCCCAA ATGCTGCTGT GGTTCAACCA GGCAAGATCATCCGGTGAAA GAACCAGTCC 4380 CTGGGCCCCA AGGATGCCGG GGAAACAGGA CCTTCTCCTTTCCTGGAGCT GGTTTAGCTG 4440 GATATGGGAG GGGGTTTGGC TGCCTGTGCC CAGGAGCTAGACTGCCTTGA GGCTGCTGTC 4500 CTTTCACAGC CATGGAGTAG AGGCCTAAGT TGACACTGCCCTGGGCAGAC AAGACAGGAG 4560 CTGTCGCCCC AGGCCTGTGC TGCCCAGCCA GGAACCCTGTACTGCTGCTG CGACCTGATG 4620 CTGCCAGTCT GTTAAAATAA AGATAAGAGA CTTGGACTCCAGACCCCTGT GTGACTGTCC 4680 CAATTTCTTC TTTCCAGGCA AGCAGGGCAA GGAGATCTTTGGAGCAAGAT CATAACTGAG 4740 G 4741 15 base pairs nucleic acid singleunknown cDNA 5 GGTTACCGTG TGTAC 15 15 base pairs nucleic acid singleunknown cDNA 6 GGTTACCTTG TGTAC 15 15 base pairs nucleic acid singleunknown DNA (genomic) CDS 1..15 7 CTG TGC CGC CAC CTG 15 Leu Cys Arg HisLeu 1 5 5 amino acids amino acid unknown protein 8 Leu Cys Arg His Leu 15 12 base pairs nucleic acid double unknown DNA (genomic) CDS 1..12 9CTG TGC CAC CTG 12 Leu Cys His Leu 1 4 amino acids amino acid unknownprotein 10 Leu Cys His Leu 1 29 base pairs nucleic acid single unknownDNA 11 AGTAGTCTCG AGACGGGACA GACGAACCA 29 31 base pairs nucleic acidsingle unknown DNA 12 AGTAGTCTGC AGAGCAGGGT ACATGGCACT G 31 29 basepairs nucleic acid single unknown DNA 13 ATCATCAAGC TTGGGTAACC ATGAAAGCA29 32 base pairs nucleic acid single unknown DNA 14 ATCATCGAATTCTACAATTC GGTAATAATT CC 32 19 base pairs nucleic acid single unknownDNA 15 CTCCACGGAT CCCGCAGGA 19 32 base pairs nucleic acid single unknownDNA 16 AGTAGTGTCG ACTTGCCTGG TTGAACCACA GC 32 27 base pairs nucleic acidsingle unknown DNA 17 AGTAGTCGAC ATGGGCAGGA TGTGTGG 27 17 base pairsnucleic acid single unknown DNA 18 CTATTTGGTA CACACGG 17 17 base pairsnucleic acid single unknown DNA 19 CTATTTGGTA CACAAGG 17 17 base pairsnucleic acid single unknown DNA 20 CTCTGTGCCG CCACCTG 17 17 base pairsnucleic acid single unknown DNA 21 GCTCTGTGCC ACCTGAT 17 28 base pairsnucleic acid single unknown DNA 22 ATCATTGAAT TCCACGGACG ATAAGTAC 28 29base pairs nucleic acid single unknown DNA 23 ATCATCCTCG AGACGGGACAGACGAACCA 29 17 base pairs nucleic acid single unknown DNA 24 GTTCCTTCTTCAGCCCG 17 27 base pairs nucleic acid single unknown cDNA 25 AGTAGTCGACTGCTAGAGCA ATCAGAG 27 23 base pairs nucleic acid single unknown DNA 26AGTGTCGACT CGTCAGGACC AAC 23 17 base pairs nucleic acid single unknownDNA 27 ATGAAGCAAT ACCTGTC 17 17 base pairs nucleic acid single unknownDNA 28 ATGAAGCAAC ACCTGTC 17 17 base pairs nucleic acid single unknownDNA 29 ACTACTCCAG GAGCTCT 17 17 base pairs nucleic acid single unknownDNA 30 ACTACTCCGG GAGCTCT 17 28 base pairs nucleic acid single unknownDNA 31 TCATCCTCGA GCACTGACCT GCACTGGG 28 17 base pairs nucleic acidsingle unknown DNA 32 GTCACAGCAC TTGTGAG 17 17 base pairs nucleic acidsingle unknown DNA 33 GTCACAGCAC CTGTGAG 17 2347 base pairs nucleic acidsingle unknown cDNA CDS 88..1974 34 GGCGCCGCCC GGGGCCCTGA GGGCTGGCTAGGGTCCAGGC CGGGGGGGAC GGGACAGACG 60 AACCAGCCCC GTGTAGGAAG CGCGACA ATGCCC CGC TAC GGA GCG TCA CTC 111 Met Pro Arg Tyr Gly Ala Ser Leu 1 5 CGCCAG AGC TGC CCC AGG TCC GGC CGG GAG CAG GGA CAA GAC GGG ACC 159 Arg GlnSer Cys Pro Arg Ser Gly Arg Glu Gln Gly Gln Asp Gly Thr 10 15 20 GCC GGAGCC CCC GGA CTC CTT TGG ATG GGC CTG GTG CTG GCG CTG GCG 207 Ala Gly AlaPro Gly Leu Leu Trp Met Gly Leu Val Leu Ala Leu Ala 25 30 35 40 CTG GCGCTG GCG CTG GCT CTG TCT GAC TCT CGG GTT CTC TGG GCT CCG 255 Leu Ala LeuAla Leu Ala Leu Ser Asp Ser Arg Val Leu Trp Ala Pro 45 50 55 GCA GAG GCTCAC CCT CTT TCT CCC CAA GGC CAT CCT GCC AGG TTA CAT 303 Ala Glu Ala HisPro Leu Ser Pro Gln Gly His Pro Ala Arg Leu His 60 65 70 CGC ATA GTG CCCCGG CTC CGA GAT GTC TTT GGG TGG GGG AAC CTC ACC 351 Arg Ile Val Pro ArgLeu Arg Asp Val Phe Gly Trp Gly Asn Leu Thr 75 80 85 TGC CCA ATC TGC AAAGGT CTA TTC ACC GCC ATC AAC CTC GGG CTG AAG 399 Cys Pro Ile Cys Lys GlyLeu Phe Thr Ala Ile Asn Leu Gly Leu Lys 90 95 100 AAG GAA CCC AAT GTGGCT CGC GTG GGC TCC GTG GCC ATC AAG CTG TGC 447 Lys Glu Pro Asn Val AlaArg Val Gly Ser Val Ala Ile Lys Leu Cys 105 110 115 120 AAT CTG CTG AAGATA GCA CCA CCT GCC GTG TGC CAA TCC ATT GTC CAC 495 Asn Leu Leu Lys IleAla Pro Pro Ala Val Cys Gln Ser Ile Val His 125 130 135 CTC TTT GAG GATGAC ATG GTG GAG GTG TGG AGA CGC TCA GTG CTG AGC 543 Leu Phe Glu Asp AspMet Val Glu Val Trp Arg Arg Ser Val Leu Ser 140 145 150 CCA TCT GAG GCCTGT GGC CTG CTC CTG GGC TCC ACC TGT GGG CAC TGG 591 Pro Ser Glu Ala CysGly Leu Leu Leu Gly Ser Thr Cys Gly His Trp 155 160 165 GAC ATT TTC TCATCT TGG AAC ATC TCT TTG CCT ACT GTG CCG AAG CCG 639 Asp Ile Phe Ser SerTrp Asn Ile Ser Leu Pro Thr Val Pro Lys Pro 170 175 180 CCC CCC AAA CCCCCT AGC CCC CCA GCC CCA GGT GCC CCT GTC AGC CGC 687 Pro Pro Lys Pro ProSer Pro Pro Ala Pro Gly Ala Pro Val Ser Arg 185 190 195 200 ATC CTC TTCCTC ACT GAC CTG CAC TGG GAT CAT GAC TAC CTG GAG GGC 735 Ile Leu Phe LeuThr Asp Leu His Trp Asp His Asp Tyr Leu Glu Gly 205 210 215 ACG GAC CCTGAC TGT GCA GAC CCA CTG TGC TGC CGC CGG GGT TCT GGC 783 Thr Asp Pro AspCys Ala Asp Pro Leu Cys Cys Arg Arg Gly Ser Gly 220 225 230 CTG CCG CCCGCA TCC CGG CCA GGT GCC GGA TAC TGG GGC GAA TAC AGC 831 Leu Pro Pro AlaSer Arg Pro Gly Ala Gly Tyr Trp Gly Glu Tyr Ser 235 240 245 AAG TGT GACCTG CCC CTG AGG ACC CTG GAG AGC CTG TTG AGT GGG CTG 879 Lys Cys Asp LeuPro Leu Arg Thr Leu Glu Ser Leu Leu Ser Gly Leu 250 255 260 GGC CCA GCCGGC CCT TTT GAT ATG GTG TAC TGG ACA GGA GAC ATC CCC 927 Gly Pro Ala GlyPro Phe Asp Met Val Tyr Trp Thr Gly Asp Ile Pro 265 270 275 280 GCA CATGAT GTC TGG CAC CAG ACT CGT CAG GAC CAA CTG CGG GCC CTG 975 Ala His AspVal Trp His Gln Thr Arg Gln Asp Gln Leu Arg Ala Leu 285 290 295 ACC ACCGTC ACA GCA CTT GTG AGG AAG TTC CTG GGG CCA GTG CCA GTG 1023 Thr Thr ValThr Ala Leu Val Arg Lys Phe Leu Gly Pro Val Pro Val 300 305 310 TAC CCTGCT GTG GGT AAC CAT GAA AGC ATA CCT GTC AAT AGC TTC CCT 1071 Tyr Pro AlaVal Gly Asn His Glu Ser Ile Pro Val Asn Ser Phe Pro 315 320 325 CCC CCCTTC ATT GAG GGC AAC CAC TCC TCC CGC TGG CTC TAT GAA GCG 1119 Pro Pro PheIle Glu Gly Asn His Ser Ser Arg Trp Leu Tyr Glu Ala 330 335 340 ATG GCCAAG GCT TGG GAG CCC TGG CTG CCT GCC GAA GCC CTG CGC ACC 1167 Met Ala LysAla Trp Glu Pro Trp Leu Pro Ala Glu Ala Leu Arg Thr 345 350 355 360 CTCAGA ATT GGG GGG TTC TAT GCT CTT TCC CCA TAC CCC GGT CTC CGC 1215 Leu ArgIle Gly Gly Phe Tyr Ala Leu Ser Pro Tyr Pro Gly Leu Arg 365 370 375 CTCATC TCT CTC AAT ATG AAT TTT TGT TCC CGT GAG AAC TTC TGG CTC 1263 Leu IleSer Leu Asn Met Asn Phe Cys Ser Arg Glu Asn Phe Trp Leu 380 385 390 TTGATC AAC TCC ACG GAT CCC GCA GGA CAG CTC CAG TGG CTG GTG GGG 1311 Leu IleAsn Ser Thr Asp Pro Ala Gly Gln Leu Gln Trp Leu Val Gly 395 400 405 GAGCTT CAG GCT GCT GAG GAT CGA GGA GAC AAA GTG CAT ATA ATT GGC 1359 Glu LeuGln Ala Ala Glu Asp Arg Gly Asp Lys Val His Ile Ile Gly 410 415 420 CACATT CCC CCA GGG CAC TGT CTG AAG AGC TGG AGC TGG AAT TAT TAC 1407 His IlePro Pro Gly His Cys Leu Lys Ser Trp Ser Trp Asn Tyr Tyr 425 430 435 440CGA ATT GTA GCC AGG TAT GAG AAC ACC CTG GCT GCT CAG TTC TTT GGC 1455 ArgIle Val Ala Arg Tyr Glu Asn Thr Leu Ala Ala Gln Phe Phe Gly 445 450 455CAC ACT CAT GTG GAT GAA TTT GAG GTC TTC TAT GAT GAA GAG ACT CTG 1503 HisThr His Val Asp Glu Phe Glu Val Phe Tyr Asp Glu Glu Thr Leu 460 465 470AGC CGG CCG CTG GCT GTA GCC TTC CTG GCA CCC AGT GCA ACT ACC TAC 1551 SerArg Pro Leu Ala Val Ala Phe Leu Ala Pro Ser Ala Thr Thr Tyr 475 480 485ATC GGC CTT AAT CCT GGT TAC CTT GTG TAC CAA ATA GAT GGA AAC TAC 1599 IleGly Leu Asn Pro Gly Tyr Leu Val Tyr Gln Ile Asp Gly Asn Tyr 490 495 500TCC AGG AGC TCT CAC GTG GTC CTG GAC CAT GAG ACC TAC ATC CTG AAT 1647 SerArg Ser Ser His Val Val Leu Asp His Glu Thr Tyr Ile Leu Asn 505 510 515520 CTG ACC CAG GCA AAC ATA CCG GGA GCC ATA CCG CAC TGG CAG CTT CTC 1695Leu Thr Gln Ala Asn Ile Pro Gly Ala Ile Pro His Trp Gln Leu Leu 525 530535 TAC AGG GCT CGA GAA ACC TAT GGG CTG CCC AAC ACA CTG CCT ACC GCC 1743Tyr Arg Ala Arg Glu Thr Tyr Gly Leu Pro Asn Thr Leu Pro Thr Ala 540 545550 TGG CAC AAC CTG GTA TAT CGC ATG CGG GGC GAC ATG CAA CTT TTC CAG 1791Trp His Asn Leu Val Tyr Arg Met Arg Gly Asp Met Gln Leu Phe Gln 555 560565 ACC TTC TGG TTT CTC TAC CAT AAG GGC CAC CCA CCC TCG GAG CCC TGT 1839Thr Phe Trp Phe Leu Tyr His Lys Gly His Pro Pro Ser Glu Pro Cys 570 575580 GGC ACG CCC TGC CGT CTG GCT ACT CTT TGT GCC CAG CTC TCT GCC CGT 1887Gly Thr Pro Cys Arg Leu Ala Thr Leu Cys Ala Gln Leu Ser Ala Arg 585 590595 600 GCT GAC AGC CCT GCT CTG TGC CGC CAC CTG ATG CCA GAT GGG AGC CTC1935 Ala Asp Ser Pro Ala Leu Cys Arg His Leu Met Pro Asp Gly Ser Leu 605610 615 CCA GAG GCC CAG AGC CTG TGG CCA AGG CCA CTG TTT TGC TAGGGCCCCA1984 Pro Glu Ala Gln Ser Leu Trp Pro Arg Pro Leu Phe Cys 620 625GGGCCCACAT TTGGGAAAGT TCTTGATGTA GGAAAGGGTG AAAAAGCCCA AATGCTGCTG 2044TGGTTCAACC AGGCAAGATC ATCCGGTGAA AGAACCAGTC CCTGGGCCCC AAGGATGCCG 2104GGGAAACAGG ACCTTCTCCT TTCCTGGAGC TGGTTTAGCT GGATATGGGA GGGGGTTTGG 2164CTGCCTGTGC CCAGGAGCTA GACTGCCTTG AGGCTGCTGT CCTTTCACAG CCATGGAGTA 2224GAGGCCTAAG TTGACACTGC CCTGGGCAGA CAAGACAGGA GCTGTCGCCC CAGGCCTGTG 2284CTGCCCAGCC AGGAACCCTG TACTGCTGCT GCGACCTGAT GCTGCCAGTC TGTTAAAATA 2344AAG 2347 2344 base pairs nucleic acid single unknown cDNA CDS 88..197135 GGCGCCGCCC GGGGCCCTGA GGGCTGGCTA GGGTCCAGGC CGGGGGGGAC GGGACAGACG 60AACCAGCCCC GTGTAGGAAG CGCGACA ATG CCC CGC TAC GGA GCG TCA CTC 111 MetPro Arg Tyr Gly Ala Ser Leu 1 5 CGC CAG AGC TGC CCC AGG TCC GGC CGG GAGCAG GGA CAA GAC GGG ACC 159 Arg Gln Ser Cys Pro Arg Ser Gly Arg Glu GlnGly Gln Asp Gly Thr 10 15 20 GCC GGA GCC CCC GGA CTC CTT TGG ATG GGC CTGGTG CTG GCG CTG GCG 207 Ala Gly Ala Pro Gly Leu Leu Trp Met Gly Leu ValLeu Ala Leu Ala 25 30 35 40 CTG GCG CTG GCG CTG GCT CTG TCT GAC TCT CGGGTT CTC TGG GCT CCG 255 Leu Ala Leu Ala Leu Ala Leu Ser Asp Ser Arg ValLeu Trp Ala Pro 45 50 55 GCA GAG GCT CAC CCT CTT TCT CCC CAA GGC CAT CCTGCC AGG TTA CAT 303 Ala Glu Ala His Pro Leu Ser Pro Gln Gly His Pro AlaArg Leu His 60 65 70 CGC ATA GTG CCC CGG CTC CGA GAT GTC TTT GGG TGG GGGAAC CTC ACC 351 Arg Ile Val Pro Arg Leu Arg Asp Val Phe Gly Trp Gly AsnLeu Thr 75 80 85 TGC CCA ATC TGC AAA GGT CTA TTC ACC GCC ATC AAC CTC GGGCTG AAG 399 Cys Pro Ile Cys Lys Gly Leu Phe Thr Ala Ile Asn Leu Gly LeuLys 90 95 100 AAG GAA CCC AAT GTG GCT CGC GTG GGC TCC GTG GCC ATC AAGCTG TGC 447 Lys Glu Pro Asn Val Ala Arg Val Gly Ser Val Ala Ile Lys LeuCys 105 110 115 120 AAT CTG CTG AAG ATA GCA CCA CCT GCC GTG TGC CAA TCCATT GTC CAC 495 Asn Leu Leu Lys Ile Ala Pro Pro Ala Val Cys Gln Ser IleVal His 125 130 135 CTC TTT GAG GAT GAC ATG GTG GAG GTG TGG AGA CGC TCAGTG CTG AGC 543 Leu Phe Glu Asp Asp Met Val Glu Val Trp Arg Arg Ser ValLeu Ser 140 145 150 CCA TCT GAG GCC TGT GGC CTG CTC CTG GGC TCC ACC TGTGGG CAC TGG 591 Pro Ser Glu Ala Cys Gly Leu Leu Leu Gly Ser Thr Cys GlyHis Trp 155 160 165 GAC ATT TTC TCA TCT TGG AAC ATC TCT TTG CCT ACT GTGCCG AAG CCG 639 Asp Ile Phe Ser Ser Trp Asn Ile Ser Leu Pro Thr Val ProLys Pro 170 175 180 CCC CCC AAA CCC CCT AGC CCC CCA GCC CCA GGT GCC CCTGTC AGC CGC 687 Pro Pro Lys Pro Pro Ser Pro Pro Ala Pro Gly Ala Pro ValSer Arg 185 190 195 200 ATC CTC TTC CTC ACT GAC CTG CAC TGG GAT CAT GACTAC CTG GAG GGC 735 Ile Leu Phe Leu Thr Asp Leu His Trp Asp His Asp TyrLeu Glu Gly 205 210 215 ACG GAC CCT GAC TGT GCA GAC CCA CTG TGC TGC CGCCGG GGT TCT GGC 783 Thr Asp Pro Asp Cys Ala Asp Pro Leu Cys Cys Arg ArgGly Ser Gly 220 225 230 CTG CCG CCC GCA TCC CGG CCA GGT GCC GGA TAC TGGGGC GAA TAC AGC 831 Leu Pro Pro Ala Ser Arg Pro Gly Ala Gly Tyr Trp GlyGlu Tyr Ser 235 240 245 AAG TGT GAC CTG CCC CTG AGG ACC CTG GAG AGC CTGTTG AGT GGG CTG 879 Lys Cys Asp Leu Pro Leu Arg Thr Leu Glu Ser Leu LeuSer Gly Leu 250 255 260 GGC CCA GCC GGC CCT TTT GAT ATG GTG TAC TGG ACAGGA GAC ATC CCC 927 Gly Pro Ala Gly Pro Phe Asp Met Val Tyr Trp Thr GlyAsp Ile Pro 265 270 275 280 GCA CAT GAT GTC TGG CAC CAG ACT CGT CAG GACCAA CTG CGG GCC CTG 975 Ala His Asp Val Trp His Gln Thr Arg Gln Asp GlnLeu Arg Ala Leu 285 290 295 ACC ACC GTC ACA GCA CTT GTG AGG AAG TTC CTGGGG CCA GTG CCA GTG 1023 Thr Thr Val Thr Ala Leu Val Arg Lys Phe Leu GlyPro Val Pro Val 300 305 310 TAC CCT GCT GTG GGT AAC CAT GAA AGC ATA CCTGTC AAT AGC TTC CCT 1071 Tyr Pro Ala Val Gly Asn His Glu Ser Ile Pro ValAsn Ser Phe Pro 315 320 325 CCC CCC TTC ATT GAG GGC AAC CAC TCC TCC CGCTGG CTC TAT GAA GCG 1119 Pro Pro Phe Ile Glu Gly Asn His Ser Ser Arg TrpLeu Tyr Glu Ala 330 335 340 ATG GCC AAG GCT TGG GAG CCC TGG CTG CCT GCCGAA GCC CTG CGC ACC 1167 Met Ala Lys Ala Trp Glu Pro Trp Leu Pro Ala GluAla Leu Arg Thr 345 350 355 360 CTC AGA ATT GGG GGG TTC TAT GCT CTT TCCCCA TAC CCC GGT CTC CGC 1215 Leu Arg Ile Gly Gly Phe Tyr Ala Leu Ser ProTyr Pro Gly Leu Arg 365 370 375 CTC ATC TCT CTC AAT ATG AAT TTT TGT TCCCGT GAG AAC TTC TGG CTC 1263 Leu Ile Ser Leu Asn Met Asn Phe Cys Ser ArgGlu Asn Phe Trp Leu 380 385 390 TTG ATC AAC TCC ACG GAT CCC GCA GGA CAGCTC CAG TGG CTG GTG GGG 1311 Leu Ile Asn Ser Thr Asp Pro Ala Gly Gln LeuGln Trp Leu Val Gly 395 400 405 GAG CTT CAG GCT GCT GAG GAT CGA GGA GACAAA GTG CAT ATA ATT GGC 1359 Glu Leu Gln Ala Ala Glu Asp Arg Gly Asp LysVal His Ile Ile Gly 410 415 420 CAC ATT CCC CCA GGG CAC TGT CTG AAG AGCTGG AGC TGG AAT TAT TAC 1407 His Ile Pro Pro Gly His Cys Leu Lys Ser TrpSer Trp Asn Tyr Tyr 425 430 435 440 CGA ATT GTA GCC AGG TAT GAG AAC ACCCTG GCT GCT CAG TTC TTT GGC 1455 Arg Ile Val Ala Arg Tyr Glu Asn Thr LeuAla Ala Gln Phe Phe Gly 445 450 455 CAC ACT CAT GTG GAT GAA TTT GAG GTCTTC TAT GAT GAA GAG ACT CTG 1503 His Thr His Val Asp Glu Phe Glu Val PheTyr Asp Glu Glu Thr Leu 460 465 470 AGC CGG CCG CTG GCT GTA GCC TTC CTGGCA CCC AGT GCA ACT ACC TAC 1551 Ser Arg Pro Leu Ala Val Ala Phe Leu AlaPro Ser Ala Thr Thr Tyr 475 480 485 ATC GGC CTT AAT CCT GGT TAC CGT GTGTAC CAA ATA GAT GGA AAC TAC 1599 Ile Gly Leu Asn Pro Gly Tyr Arg Val TyrGln Ile Asp Gly Asn Tyr 490 495 500 TCC AGG AGC TCT CAC GTG GTC CTG GACCAT GAG ACC TAC ATC CTG AAT 1647 Ser Arg Ser Ser His Val Val Leu Asp HisGlu Thr Tyr Ile Leu Asn 505 510 515 520 CTG ACC CAG GCA AAC ATA CCG GGAGCC ATA CCG CAC TGG CAG CTT CTC 1695 Leu Thr Gln Ala Asn Ile Pro Gly AlaIle Pro His Trp Gln Leu Leu 525 530 535 TAC AGG GCT CGA GAA ACC TAT GGGCTG CCC AAC ACA CTG CCT ACC GCC 1743 Tyr Arg Ala Arg Glu Thr Tyr Gly LeuPro Asn Thr Leu Pro Thr Ala 540 545 550 TGG CAC AAC CTG GTA TAT CGC ATGCGG GGC GAC ATG CAA CTT TTC CAG 1791 Trp His Asn Leu Val Tyr Arg Met ArgGly Asp Met Gln Leu Phe Gln 555 560 565 ACC TTC TGG TTT CTC TAC CAT AAGGGC CAC CCA CCC TCG GAG CCC TGT 1839 Thr Phe Trp Phe Leu Tyr His Lys GlyHis Pro Pro Ser Glu Pro Cys 570 575 580 GGC ACG CCC TGC CGT CTG GCT ACTCTT TGT GCC CAG CTC TCT GCC CGT 1887 Gly Thr Pro Cys Arg Leu Ala Thr LeuCys Ala Gln Leu Ser Ala Arg 585 590 595 600 GCT GAC AGC CCT GCT CTG TGCCAC CTG ATG CCA GAT GGG AGC CTC CCA 1935 Ala Asp Ser Pro Ala Leu Cys HisLeu Met Pro Asp Gly Ser Leu Pro 605 610 615 GAG GCC CAG AGC CTG TGG CCAAGG CCA CTG TTT TGC TAGGGCCCCA 1981 Glu Ala Gln Ser Leu Trp Pro Arg ProLeu Phe Cys 620 625 GGGCCCACAT TTGGGAAAGT TCTTGATGTA GGAAAGGGTGAAAAAGCCCA AATGCTGCTG 2041 TGGTTCAACC AGGCAAGATC ATCCGGTGAA AGAACCAGTCCCTGGGCCCC AAGGATGCCG 2101 GGGAAACAGG ACCTTCTCCT TTCCTGGAGC TGGTTTAGCTGGATATGGGA GGGGGTTTGG 2161 CTGCCTGTGC CCAGGAGCTA GACTGCCTTG AGGCTGCTGTCCTTTCACAG CCATGGAGTA 2221 GAGGCCTAAG TTGACACTGC CCTGGGCAGA CAAGACAGGAGCTGTCGCCC CAGGCCTGTG 2281 CTGCCCAGCC AGGAACCCTG TACTGCTGCT GCGACCTGATGCTGCCAGTC TGTTAAAATA 2341 AAG 2344 2347 base pairs nucleic acid singleunknown cDNA CDS 88..1974 36 GGCGCCGCCC GGGGCCCTGA GGGCTGGCTA GGGTCCAGGCCGGGGGGGAC GGGACAGACG 60 AACCAGCCCC GTGTAGGAAG CGCGACA ATG CCC CGC TACGGA GCG TCA CTC 111 Met Pro Arg Tyr Gly Ala Ser Leu 1 5 CGC CAG AGC TGCCCC AGG TCC GGC CGG GAG CAG GGA CAA GAC GGG ACC 159 Arg Gln Ser Cys ProArg Ser Gly Arg Glu Gln Gly Gln Asp Gly Thr 10 15 20 GCC GGA GCC CCC GGACTC CTT TGG ATG GGC CTG GTG CTG GCG CTG GCG 207 Ala Gly Ala Pro Gly LeuLeu Trp Met Gly Leu Val Leu Ala Leu Ala 25 30 35 40 CTG GCG CTG GCG CTGGCT CTG TCT GAC TCT CGG GTT CTC TGG GCT CCG 255 Leu Ala Leu Ala Leu AlaLeu Ser Asp Ser Arg Val Leu Trp Ala Pro 45 50 55 GCA GAG GCT CAC CCT CTTTCT CCC CAA GGC CAT CCT GCC AGG TTA CAT 303 Ala Glu Ala His Pro Leu SerPro Gln Gly His Pro Ala Arg Leu His 60 65 70 CGC ATA GTG CCC CGG CTC CGAGAT GTC TTT GGG TGG GGG AAC CTC ACC 351 Arg Ile Val Pro Arg Leu Arg AspVal Phe Gly Trp Gly Asn Leu Thr 75 80 85 TGC CCA ATC TGC AAA GGT CTA TTCACC GCC ATC AAC CTC GGG CTG AAG 399 Cys Pro Ile Cys Lys Gly Leu Phe ThrAla Ile Asn Leu Gly Leu Lys 90 95 100 AAG GAA CCC AAT GTG GCT CGC GTGGGC TCC GTG GCC ATC AAG CTG TGC 447 Lys Glu Pro Asn Val Ala Arg Val GlySer Val Ala Ile Lys Leu Cys 105 110 115 120 AAT CTG CTG AAG ATA GCA CCACCT GCC GTG TGC CAA TCC ATT GTC CAC 495 Asn Leu Leu Lys Ile Ala Pro ProAla Val Cys Gln Ser Ile Val His 125 130 135 CTC TTT GAG GAT GAC ATG GTGGAG GTG TGG AGA CGC TCA GTG CTG AGC 543 Leu Phe Glu Asp Asp Met Val GluVal Trp Arg Arg Ser Val Leu Ser 140 145 150 CCA TCT GAG GCC TGT GGC CTGCTC CTG GGC TCC ACC TGT GGG CAC TGG 591 Pro Ser Glu Ala Cys Gly Leu LeuLeu Gly Ser Thr Cys Gly His Trp 155 160 165 GAC ATT TTC TCA TCT TGG AACATC TCT TTG CCT ACT GTG CCG AAG CCG 639 Asp Ile Phe Ser Ser Trp Asn IleSer Leu Pro Thr Val Pro Lys Pro 170 175 180 CCC CCC AAA CCC CCT AGC CCCCCA GCC CCA GGT GCC CCT GTC AGC CGC 687 Pro Pro Lys Pro Pro Ser Pro ProAla Pro Gly Ala Pro Val Ser Arg 185 190 195 200 ATC CTC TTC CTC ACT GACCTG CAC TGG GAT CAT GAC TAC CTG GAG GGC 735 Ile Leu Phe Leu Thr Asp LeuHis Trp Asp His Asp Tyr Leu Glu Gly 205 210 215 ACG GAC CCT GAC TGT GCAGAC CCA CTG TGC TGC CGC CGG GGT TCT GGC 783 Thr Asp Pro Asp Cys Ala AspPro Leu Cys Cys Arg Arg Gly Ser Gly 220 225 230 CTG CCG CCC GCA TCC CGGCCA GGT GCC GGA TAC TGG GGC GAA TAC AGC 831 Leu Pro Pro Ala Ser Arg ProGly Ala Gly Tyr Trp Gly Glu Tyr Ser 235 240 245 AAG TGT GAC CTG CCC CTGAGG ACC CTG GAG AGC CTG TTG AGT GGG CTG 879 Lys Cys Asp Leu Pro Leu ArgThr Leu Glu Ser Leu Leu Ser Gly Leu 250 255 260 GGC CCA GCC GGC CCT TTTGAT ATG GTG TAC TGG ACA GGA GAC ATC CCC 927 Gly Pro Ala Gly Pro Phe AspMet Val Tyr Trp Thr Gly Asp Ile Pro 265 270 275 280 GCA CAT GAT GTC TGGCAC CAG ACT CGT CAG GAC CAA CTG CGG GCC CTG 975 Ala His Asp Val Trp HisGln Thr Arg Gln Asp Gln Leu Arg Ala Leu 285 290 295 ACC ACC GTC ACA GCACCT GTG AGG AAG TTC CTG GGG CCA GTG CCA GTG 1023 Thr Thr Val Thr Ala ProVal Arg Lys Phe Leu Gly Pro Val Pro Val 300 305 310 TAC CCT GCT GTG GGTAAC CAT GAA AGC ATA CCT GTC AAT AGC TTC CCT 1071 Tyr Pro Ala Val Gly AsnHis Glu Ser Ile Pro Val Asn Ser Phe Pro 315 320 325 CCC CCC TTC ATT GAGGGC AAC CAC TCC TCC CGC TGG CTC TAT GAA GCG 1119 Pro Pro Phe Ile Glu GlyAsn His Ser Ser Arg Trp Leu Tyr Glu Ala 330 335 340 ATG GCC AAG GCT TGGGAG CCC TGG CTG CCT GCC GAA GCC CTG CGC ACC 1167 Met Ala Lys Ala Trp GluPro Trp Leu Pro Ala Glu Ala Leu Arg Thr 345 350 355 360 CTC AGA ATT GGGGGG TTC TAT GCT CTT TCC CCA TAC CCC GGT CTC CGC 1215 Leu Arg Ile Gly GlyPhe Tyr Ala Leu Ser Pro Tyr Pro Gly Leu Arg 365 370 375 CTC ATC TCT CTCAAT ATG AAT TTT TGT TCC CGT GAG AAC TTC TGG CTC 1263 Leu Ile Ser Leu AsnMet Asn Phe Cys Ser Arg Glu Asn Phe Trp Leu 380 385 390 TTG ATC AAC TCCACG GAT CCC GCA GGA CAG CTC CAG TGG CTG GTG GGG 1311 Leu Ile Asn Ser ThrAsp Pro Ala Gly Gln Leu Gln Trp Leu Val Gly 395 400 405 GAG CTT CAG GCTGCT GAG GAT CGA GGA GAC AAA GTG CAT ATA ATT GGC 1359 Glu Leu Gln Ala AlaGlu Asp Arg Gly Asp Lys Val His Ile Ile Gly 410 415 420 CAC ATT CCC CCAGGG CAC TGT CTG AAG AGC TGG AGC TGG AAT TAT TAC 1407 His Ile Pro Pro GlyHis Cys Leu Lys Ser Trp Ser Trp Asn Tyr Tyr 425 430 435 440 CGA ATT GTAGCC AGG TAT GAG AAC ACC CTG GCT GCT CAG TTC TTT GGC 1455 Arg Ile Val AlaArg Tyr Glu Asn Thr Leu Ala Ala Gln Phe Phe Gly 445 450 455 CAC ACT CATGTG GAT GAA TTT GAG GTC TTC TAT GAT GAA GAG ACT CTG 1503 His Thr His ValAsp Glu Phe Glu Val Phe Tyr Asp Glu Glu Thr Leu 460 465 470 AGC CGG CCGCTG GCT GTA GCC TTC CTG GCA CCC AGT GCA ACT ACC TAC 1551 Ser Arg Pro LeuAla Val Ala Phe Leu Ala Pro Ser Ala Thr Thr Tyr 475 480 485 ATC GGC CTTAAT CCT GGT TAC CGT GTG TAC CAA ATA GAT GGA AAC TAC 1599 Ile Gly Leu AsnPro Gly Tyr Arg Val Tyr Gln Ile Asp Gly Asn Tyr 490 495 500 TCC AGG AGCTCT CAC GTG GTC CTG GAC CAT GAG ACC TAC ATC CTG AAT 1647 Ser Arg Ser SerHis Val Val Leu Asp His Glu Thr Tyr Ile Leu Asn 505 510 515 520 CTG ACCCAG GCA AAC ATA CCG GGA GCC ATA CCG CAC TGG CAG CTT CTC 1695 Leu Thr GlnAla Asn Ile Pro Gly Ala Ile Pro His Trp Gln Leu Leu 525 530 535 TAC AGGGCT CGA GAA ACC TAT GGG CTG CCC AAC ACA CTG CCT ACC GCC 1743 Tyr Arg AlaArg Glu Thr Tyr Gly Leu Pro Asn Thr Leu Pro Thr Ala 540 545 550 TGG CACAAC CTG GTA TAT CGC ATG CGG GGC GAC ATG CAA CTT TTC CAG 1791 Trp His AsnLeu Val Tyr Arg Met Arg Gly Asp Met Gln Leu Phe Gln 555 560 565 ACC TTCTGG TTT CTC TAC CAT AAG GGC CAC CCA CCC TCG GAG CCC TGT 1839 Thr Phe TrpPhe Leu Tyr His Lys Gly His Pro Pro Ser Glu Pro Cys 570 575 580 GGC ACGCCC TGC CGT CTG GCT ACT CTT TGT GCC CAG CTC TCT GCC CGT 1887 Gly Thr ProCys Arg Leu Ala Thr Leu Cys Ala Gln Leu Ser Ala Arg 585 590 595 600 GCTGAC AGC CCT GCT CTG TGC CGC CAC CTG ATG CCA GAT GGG AGC CTC 1935 Ala AspSer Pro Ala Leu Cys Arg His Leu Met Pro Asp Gly Ser Leu 605 610 615 CCAGAG GCC CAG AGC CTG TGG CCA AGG CCA CTG TTT TGC TAGGGCCCCA 1984 Pro GluAla Gln Ser Leu Trp Pro Arg Pro Leu Phe Cys 620 625 GGGCCCACATTTGGGAAAGT TCTTGATGTA GGAAAGGGTG AAAAAGCCCA AATGCTGCTG 2044 TGGTTCAACCAGGCAAGATC ATCCGGTGAA AGAACCAGTC CCTGGGCCCC AAGGATGCCG 2104 GGGAAACAGGACCTTCTCCT TTCCTGGAGC TGGTTTAGCT GGATATGGGA GGGGGTTTGG 2164 CTGCCTGTGCCCAGGAGCTA GACTGCCTTG AGGCTGCTGT CCTTTCACAG CCATGGAGTA 2224 GAGGCCTAAGTTGACACTGC CCTGGGCAGA CAAGACAGGA GCTGTCGCCC CAGGCCTGTG 2284 CTGCCCAGCCAGGAACCCTG TACTGCTGCT GCGACCTGAT GCTGCCAGTC TGTTAAAATA 2344 AAG 2347

We claim:
 1. An isolated acid sphingomyelinase polypeptide comprisingSEQ ID NO: 2, wherein said polypeptide possesses ASM activity.
 2. Anisolated human acid sphingomyelinase polypeptide encoded by a nucleicacid which specifically hybridizes, under stringent conditions, to anucleic acid having a nucleotide sequence as depicted in FIG. 3 (SEQ IDNO: 1) or FIG. 6B (SEQ ID NO:4), wherein said polypeptide possesses ASMactivity.
 3. A method of producing an isolated human acidsphingomyelinase polypeptide comprising the amino acid sequence of SEQID NO:2, comprising: (a) incubating a cell comprising a nucleic acidwhich encodes a functionally active human acid sphingomyelinase proteinhaving an amino acid sequence of SEQ ID NO:2, under conditions whereinthe nucleic acid sequence is expressed and the acid sphingomyelinase isproduced; and (b) recovering the acid sphingomyelinase.
 4. A method fortreating a patient with Type B Niemann-Pick Disease comprisingadministering to the patient a therapeutically effective dose of arecombinant human acid sphingomyelinase or an isolated human acidsphingomyelinase comprising the amino acid sequence of SEQ ID NO: 2, ora recombinant variant thereof encoded by a nucleic acid whichspecifically hybridizes, under stringent conditions, to a nucleic acidhaving a nucleotide sequence as depicted in FIG. 3 (SEQ ID NO: 1) orFIG. 6B (SEQ ID NO: 4), wherein said variant possesses ASM activity. 5.The method of claim 4 wherein the therapeutically effective dose rangesfrom about 0.1 μg/kg body weight to about 10 mg/kg body weight.
 6. Themethod of claim 4 wherein the therapeutically effective dose ranges fromabout 0.1 mg/kg body weight to about 2 mg/kg body weight.
 7. The methodof claim 4 wherein the recombinant acid sphingomyelinase or therecombinant variant is administered.
 8. The method of claim 4 whereinthe isolated human acid sphingomyelinase is administered.
 9. An isolatedhuman acid sphingomyelinase polypeptide of SEQ ID NO:2 comprising atleast one silent amino acid change, wherein said polypeptide possessesASM activity.
 10. An isolated human acid sphingomyelinase polypeptide ofSEQ ID NO:2 comprising at least one silent amino acid change, whereinthe silent amino acid change is a deletion of at least one residue andsaid polypeptide possesses ASM activity.
 11. An isolated human acidsphingomyelinase polypeptide of SEQ ID NO:2 comprising at least onesilent amino acid change, wherein the silent amino acid change is anaddition of at least one residue and said polypeptide possesses ASMactivity.
 12. An isolated human acid sphingomyelinase polypeptide of SEQID NO:2 comprising at least one silent amino acid change, wherein thesilent amino acid change is a substitution of at least one residue andsaid polypeptide possesses ASM activity.
 13. An isolated human acidsphingomyelinase polypeptide comprising SEQ ID NO: 2, wherein saidpolypeptide possesses ASM activity.
 14. An isolated human acidsphingomyelinase polypeptide consisting essentially of SEQ ID NO: 2,wherein said polypeptide possesses ASM activity.
 15. An isolated humanacid sphingomyelinase polypeptide consisting of SEQ ID NO: 2, whereinsaid polypeptide possesses ASM activity.
 16. An isolated human acidsphingomyelinase polypeptide comprising SEQ ID NO:
 2. 17. An isolatedhuman acid sphingomyelinase polypeptide consisting essentially of SEQ IDNO:
 2. 18. An isolated human acid sphingomyelinase polypeptideconsisting of SEQ ID NO: 2.